Short peptides derived from pigment epithelium-derived factor attenuate retinal ischemia reperfusion injury through inhibition of apoptosis and inflammatory response in rats.

Pigment epithelium-derived factor (PEDF) is widely recognized as a neuroprotective factor expressed in the retina and has shown therapeutic potential in several retinal diseases. Our study aimed to identify the neuroprotective fragment in PEDF and investigate its protective activity in retinas under ischemia-reperfusion (IR) condition. We synthesized a series of shorter synthetic peptides, 6-mer (Ser93-Gln98) and its d-form variant (6 dS) derived from the 44-mer (Val78-Thr121; a PEDF neurotrophic fragment), to determine their cytoprotective activity in IR injury, which was induced in rat retinas by injection of saline into the anterior chamber to increase the intraocular pressure (IOP) followed by reperfusion. We found the cytoprotective effect of 6-mer on glutamate-treated Neuro-2a cells and tert-butyl hydroperoxide (tBHP)-treated 661W cells were 2.6-fold and 1.5-fold higher than the 44-mer, respectively. The cytoprotective effect was blocked by a chemical inhibitor atglistatin and blocking antibody targeting PEDF receptor (PEDF-R). IR induced several impairments in retina, including cell apoptosis, activation of microglia/macroglia, degeneration of retinal capillaries, reduction in electroretinography (ERG) amplitudes, and retinal atrophy. Such IR injuries were ameliorated by treatment with 6-mer and 6 dS eye drops. Also, the neuroprotective activity of 6-mer and 6 dS in ischemic retinas were dramatically reversed by atglistatin preconditioning. Taken together, our data demonstrate smallest neuroprotective fragment of PEDF has potential to treat retinal degeneration-related diseases.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death.

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1-7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

Mesencephalic astrocyte-derived neurotrophic factor (MANF): Structure, functions and therapeutic potential.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel evolutionarily conserved protein present in both vertebrate and invertebrate species. MANF shows distinct structural and functional properties than the traditional neurotrophic factors (NTF). MANF is composed of an N-terminal saposin-like lipid-binding domain and a C-terminal SAF-A/B, Acinus and PIAS (SAP) domain connected by a short linker. The two well-described activities of MANF include (1) role as a neurotrophic factor that plays direct neuroprotective effects in the nervous system and (2) cell protective effects in the animal models of non-neuronal diseases, including retinal damage, diabetes mellitus, liver injury, myocardial infarction, nephrotic syndrome, etc. The main objective of the current review is to provide up-to-date insights regarding the structure of MANF, mechanisms regulating its expression and secretion, physiological functions in various tissues and organs, protective effects during aging, and potential clinical applications. Together, this review highlights the importance of MANF in reversing age-related dysfunction and geroprotection.

Crystal structure of Ankyrin-G in complex with a fragment of Neurofascin reveals binding mechanisms required for integrity of the axon initial segment.

The axon initial segment (AIS) has characteristically dense clustering of voltage-gated sodium channels (Nav), cell adhesion molecule Neurofascin 186 (Nfasc), and neuronal scaffold protein Ankyrin-G (AnkG) in neurons, which facilitates generation of an action potential and maintenance of axonal polarity. However, the mechanisms underlying AIS assembly, maintenance, and plasticity remain poorly understood. Here, we report the high-resolution crystal structure of the AnkG ankyrin repeat (ANK repeat) domain in complex with its binding site in the Nfasc cytoplasmic tail that shows, in conjunction with binding affinity assays with serial truncation variants, the molecular basis of AnkG-Nfasc binding. We confirm AnkG interacts with the FIGQY motif in Nfasc, and we identify another region required for their high affinity binding. Our structural analysis revealed that ANK repeats form 4 hydrophobic or hydrophilic layers in the AnkG inner groove that coordinate interactions with essential Nfasc residues, including F1202, E1204, and Y1212. Moreover, we show disruption of the AnkG-Nfasc complex abolishes Nfasc enrichment at the AIS in cultured mouse hippocampal neurons. Finally, our structural and biochemical analysis indicated that L1 syndrome-associated mutations in L1CAM, a member of the L1 immunoglobulin family proteins including Nfasc, L1CAM, NrCAM, and CHL1, compromise binding with ankyrins. Taken together, these results define the mechanisms underlying AnkG-Nfasc complex formation and show that AnkG-dependent clustering of Nfasc is required for AIS integrity.

[Synthetic Studies on Small Molecule Natural Products with Neurotrophic Activity].

Neurotrophic factors have been shown to potentially be beneficial for the treatment of neurodegenerative diseases such as Alzheimer's disease, because endogenous neurotrophic factors (NGF, BDNF) have been recognized to play critical roles in the promotion of neurogenesis, differentiation, and neuroprotection throughout the development of the central nervous system. However, high-molecular-weight proteins are unable to cross the blood-brain barrier and are easily decomposed under physiological conditions. Thus, small molecules that can mimic the functions of neurotrophic factors are promising alternatives for the treatment of neurodegenerative disease. Since 1990, the author has been involved in searching for natural products with typical neurotrophic properties that can cause neurogenesis, enhance neurite outgrowth, and protect against neuronal death by using three cellular systems (PC12, rat cortical neurons, and MEB5 cells). Through these research activities on neurotrophic natural products, the author has tried to induce a paradigm shift from the discipline of natural products chemistry to science disciplines. This review focuses on our independent synthetic studies of the neurotrophic natural products discovered in the plants. The following synthetic elaborations are described: syntheses of dimeric isocuparane-type sesquiterpenes mastigophorenes A and B, macrocyclic bis-bibenzyls plagiochins A-D and cavicularin through a Pd-catalyzed Stille-Kelly reaction; the formal synthesis of merrilactone A and jiadifenin, which are seco-prezizaane-type sesquiterpenes, through intramolecular Pd-catalyzed Mizoroki-Heck and Tsuji-Trost reactions; and finally the first enantioselective synthesis of neovibsanin B, a vibsane-type diterpene, through a Pd-catalyzed cyclic carbopalladation-carbonyl tandem reaction.

Semisynthesis and neurotrophic activity studies of novel neomajucin/majucin derivatives as neurotrophin small molecule mimetics.

Majucin-type Illicium sesquiterpenes with potent neurotrophic activity are considered to be promising candidates for the treatment of various neurodegenerative disease. Owing to the low-abundance metabolites in Illicium genus, there are few studies on their structural modifications, structure-activity relationships, and pharmacophoric motif. Herein, structural modifications were conducted on the hydroxyl groups at C-3 and C-6 positions of two majucin-type compounds neomajucin (1) and majucin (2), and 39 neomajucin/majucin based esters were synthesized and evaluated for their neurite outgrowth-promoting activities. Among all the target derivatives, compounds 1a, 1j, 1r, 2b, 2d, 3a, 3b, 3d and 3h displayed more potent neurite outgrowth-promoting activity than their precursors. Some interesting structure-activity relationships (SARs) were also observed. Moreover, compound 1a showed good neuroprotective effect on MPP+-induced PC12 cell damage. Finally, compounds 1a and 3a exhibited relatively no cytotoxicity to normal human H9C2 cardiac cells. This work will shed light on the development of neomajucin/majucin derivatives as potential neurotrophic agents.

Cerebral dopamine neurotrophic factor reduces α-synuclein aggregation and propagation and alleviates behavioral alterations in vivo.

A molecular hallmark in Parkinson's disease (PD) pathogenesis are α-synuclein aggregates. Cerebral dopamine neurotrophic factor (CDNF) is an atypical growth factor that is mostly resident in the endoplasmic reticulum but exerts its effects both intracellularly and extracellularly. One of the beneficial effects of CDNF can be protecting neurons from the toxic effects of α-synuclein. Here, we investigated the effects of CDNF on α-synuclein aggregation in vitro and in vivo. We found that CDNF directly interacts with α-synuclein with a KD = 23 ± 6 nM and reduces its auto-association. Using nuclear magnetic resonance (NMR) spectroscopy, we identified interaction sites on the CDNF protein. Remarkably, CDNF reduces the neuronal internalization of α-synuclein fibrils and induces the formation of insoluble phosphorylated α-synuclein inclusions. Intra-striatal CDNF administration alleviates motor deficits in rodents challenged with α-synuclein fibrils, though it did not reduce the number of phosphorylated α-synuclein inclusions in the substantia nigra. CDNF's beneficial effects on rodent behavior appear not to be related to the number of inclusions formed in the current context, and further study of its effects on the aggregation mechanism in vivo are needed. Nonetheless, the interaction of CDNF with α-synuclein, modifying its aggregation, spreading, and associated behavioral alterations, provides novel insights into the potential of CDNF as a therapeutic strategy in PD and other synucleinopathies.

Neuromodulator release in neurons requires two functionally redundant calcium sensors.

Neuropeptides and neurotrophic factors secreted from dense core vesicles (DCVs) control many brain functions, but the calcium sensors that trigger their secretion remain unknown. Here, we show that in mouse hippocampal neurons, DCV fusion is strongly and equally reduced in synaptotagmin-1 (Syt1)- or Syt7-deficient neurons, but combined Syt1/Syt7 deficiency did not reduce fusion further. Cross-rescue, expression of Syt1 in Syt7-deficient neurons, or vice versa, completely restored fusion. Hence, both sensors are rate limiting, operating in a single pathway. Overexpression of either sensor in wild-type neurons confirmed this and increased fusion. Syt1 traveled with DCVs and was present on fusing DCVs, but Syt7 supported fusion largely from other locations. Finally, the duration of single DCV fusion events was reduced in Syt1-deficient but not Syt7-deficient neurons. In conclusion, two functionally redundant calcium sensors drive neuromodulator secretion in an expression-dependent manner. In addition, Syt1 has a unique role in regulating fusion pore duration.

NINJ1 mediates plasma membrane rupture during lytic cell death.

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

Design and Development of a Behaviorally Active Recombinant Neurotrophic Factor.

INTRODUCTION: Carbamoylated erythropoietin (CEPO) is a chemically engineered, nonhematopoietic derivative of erythropoietin (EPO) that retains its antidepressant and pro-cognitive effects, which are attributed to the increased expression of neurotrophic factors like brain derived neurotrophic factor (BDNF), in the central nervous system. However, the chemical modification process which produces CEPO from erythropoietin (EPO) requires pure EPO as raw material, is challenging to scale-up and can also cause batch-to-batch variability. To address these key limitations while retaining its behavioral effects, we designed, expressed and analyzed a triple, glutamine, substitution recombinant mimetic of CEPO, named QPO. METHODS AND MATERIALS: We employ a combination of computational structural biology, molecular, cellular and behavioral assays to design, produce, purify and test QPO. RESULTS: QPO was shown to be a nonhematopoietic polypeptide with significant antidepressant-like and pro-cognitive behavioral effects in rodent assays while significantly upregulating BDNF expression in-vitro and in-vivo. The in-silico binding affinity analysis of QPO bound to the EPOR/EPOR homodimer receptor shows significantly decreased binding to Active Site 2, but not Active Site 1, of EPOR. DISCUSSION: The results of the behavioral and gene expression analysis imply that QPO is a successful CEPO mimetic protein and potentially acts via a similar neurotrophic mechanism, making it a drug development target for psychiatric disorders. The decreased binding to Active Site 2 could imply that this active site is not involved in neuroactive signaling and could allow the development of a functional innate repair receptor (IRR) model. Substituting the three glutamine substitution residues with arginine (RPO) resulted in the loss of behavioral activity, indicating the importance of glutamine residues at those positions.

Cage-Monoterpenoid Quinoline Alkaloids with Neurite Growth Promoting Effects from the Fruits of Melodinus yunnanensis.

Meloyunnanines A-C, three alkaloids with an unprecedented skeleton, were isolated from fruits of Melodinus yunnanensis. The structures featuring a caged-6/6/5/6/5/5 ring system were elucidated by the analysis of comprehensive spectroscopic and X-ray data. Biosynthetically, meloyunnanines A-C were assigned to monoterpenoid quinoline alkaloids (MQAs), derived from monoterpenoid indole alkaloids through oxidation and rearrangement. These compounds together with three known Melodinus MQAs were evaluated for their neurotrophic activity and scandine N4-oxide exhibited significant effect.

Spatiotemporal regulation of PEDF signaling by type I collagen remodeling.

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.

Human VGF-Derived Antidepressant Neuropeptide TLQP62 Promotes SH-SY5Y Neurite Outgrowth.

TLQP62 is a neuropeptide derived from the neurotrophin-inducible VGF (non-acronymic) protein with antidepressant-like properties capable of inducing increased memory on the mouse hippocampus by promoting neurogenesis and synaptic plasticity through brain-derived neurotropic factor (BDNF) and its receptor tyrosine receptor kinase B (TrkB). Human SH-SY5Y neuroblastoma-derived cell line is widely used in neuroscience research and is known to undergo neurodifferentiation in the presence of all-trans retinoic acid by upregulating the expression of TrkB, making cells responsive to BDNF. As TLQP62 promotes BDNF expression, which in turn activates a BDNF/TrkB/CREB (cAMP response element-binding protein) pathway that upregulates VGF expression, there is a VGF-BDNF regulatory loop that seems to regulate neurogenesis. Therefore, here, we evaluate by morphological observation the ability of human TLQP62 to induce neuritogenesis of human SH-SY5Y neuroblastoma-derived cell line in a retinoic acid and BDFN-like way, making this cell line a suitable cell model for further studies concerning TLQP62 molecular mechanisms and signalling pathways. SIGNIFICANCE STATEMENT: VGF has been widely explored for its role in emotional behaviour and neuropsychiatric illness (Bartolomucci et al. 2011). Although VGF levels were found reduced in leukocytes of depressed patients, after antidepressant treatment or voluntary exercise, those levels were found to be restored in the hippocampus (Hunsberger et al. 2007; Thakker-Varia et al. 2007). Administration to hippocampal cells of TLQP62 produced an increase in synaptic charge that could explain this antidepressants effects (Alder et al. 2003). This interesting role of TLQP62 in the brain, especially in the hippocampus, makes this neuropeptide an attractive target for further investigation of its role in neurogenesis, learning, memory, and neurological disorders, and possible treatment development. Thus, the identification of a receptor(s) for this peptide and associated signalling pathway(s) is of high importance, as well as a proper cell model to perform those studies.

Bacterial Phytochrome as a Scaffold for Engineering of Receptor Tyrosine Kinases Controlled with Near-Infrared Light.

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.

Nanofibrous Nerve Conduits with Nerve Growth Factors and Bone Marrow Stromal Cells Pre-Cultured in Bioreactors for Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) was the leading cause of permanent dysfunction in movement and sensation. Synthesized nerve guide conduits (NGCs) with Schwann Cells (SCs) can help peripheral nerve regeneration. However, poor accessibility of SCs and lack of full coverage of seeded cells on NGCs can lead to failure of nerve regeneration across long gaps and full functional recovery. To overcome these limitations, bone marrow stromal cells (BMSCs) and a novel culture method were proposed in the current study. BMSCs were harvested and seeded on a never growth factor (NGF)-loaded PCL nanofibrous NGCs and cultured with a rotary cell culture system (RCCS) before implantation. The NGCs were tested in vitro with PC-12 cells to validate the bioactivity of released NGF and to access its ability to promote neurite extension. Also, the NGCs were tested in vivo with rat sciatic nerve model to exam its potential in bridging the long gap (15 mm segmental defect). The efficacy of the NGCs was investigated based on the results of the functional test, electrophysiology test, muscle atrophy, and histological analysis. The results of in vitro PC-12 cell study confirmed the bioactivity of released NGF and showed a significant increase in the neurite extension with the help of PEG-diamine and BSA. These results showed that the novel loading method could preserve the bioactivity of growth factors and achieve a sustained release in vitro. Besides, the results of the in vivo study exhibited a significant increase with the combination of all additives. These results showed that with the help of NGF and RCCS, the NGCs with the seeded BMSCs could enhance peripheral nerve regeneration across long nerve injury gaps.

A chitosan-based thermosensitive scaffold loaded with bone marrow-derived mesenchymal stem cells promotes motor function recovery in spinal cord injured mice.

Spinal cord injury is a devastating trauma with high mortality and disability, for which there is no effective treatment. Stem cell-based tissue engineering has been reported to promote functional neural recovery. At present, building a neural scaffold with excellent biocompatibility for cells and tissues is still challenging. In this study, a new thermosensitive composite hydrogel based on chitosan, hydroxyethyl cellulose, collagen and ß-phosphoglycerate (CS-HEC-Col/GP hydrogel) is developed to encapsulate murine bone marrow-derived mesenchymal stem cells (BMSCs) to improve therapeutic efficacy in spinal cord injured mice. This composite hydrogel possesses a good cytocompatibility to mouse BMSCs by live/dead staining, minimized inflammatory reaction in vivo by hematoxylin and eosin staining and suitable rheological behavior similar to neural tissue, ranging from 100 to 1000 Pa. Furthermore, the data from animal experiments indicated that BMSC-loaded CS-HEC-Col/GP hydrogel could enhance the survival or proliferation of endogenous nerve cells, probably by secreting neurotrophic factors and inhibiting apoptosis, and thereby promote the recovery of motor function in the hindlimbs of a murine spinal cord injury model.

Proteolytic Processing of Neuregulin 2.

Neuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.

Upregulation of VEGF and PEDF in Placentas of Women with Lower Extremity Venous Insufficiency during Pregnancy and Its Implication in Villous Calcification.

Pregnancy is a period in a woman's life in which changes can occur that affect different physiological processes. Common conditions during this period include vascular changes, such as lower extremity venous insufficiency (VI). This is an observational, analytical, and prospective cohort study in which 114 pregnant women were analyzed, of which 62 were clinically diagnosed with VI. In parallel, 52 control patients without VI (HC) were studied. The aim of this study was to observe changes in angiogenesis and inflammation markers as well as the presence of calcium deposits. The expression of vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), and pigment epithelium-derived factor (PEDF) was analyzed by immunohistochemistry and RT-qPCR. The presence of calcium deposits was revealed using the von Kossa method. In the placentas of mothers with VI, gene expression of VEGF (34.575 [32.380-36.720] VI vs 32.965 [30.580-36.320] HC) and PEDF (25.417 [24.459-27.675] VI vs 24.400 [23.102-30.223] HC) significantly increased, as was protein expression in the placental villi. An increase in calcium deposits was observed in the placentas of women with VI (72.58% VI/53.84% HC). This study revealed the existence of cellular damage in the placental villi of mothers with VI with tissue implications such as increased calcification.

In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGF Serine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve growth factor (NGF) is an endogenously produced polypeptide that promotes the differentiation, survival, and repair of neurons in the central and peripheral nervous systems. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood-brain barrier, and peripheral side effects. Previously, we found that an extract of the Momordica cochinchinensis seed stimulated PC-12 neurite outgrowth. Bioactivity-guided fractioning of the seed extract suggested that the NGF mimetic agent was one of few defined proteins from this plant: one group being the defense Knottins and the other group of the lowest mass is the potent trypsin inhibitor MCoTI-II. Here, the NGF mimetic potential of this latter protein was investigated using two concurrent but different approaches. A biological study used recombinant purified MCoTI-II, which when tested in rat PC-12 cells grown on collagen, failed to initiate outgrowth relative to the positive control 7S NGF. In a separate computational study, the possibility was investigated such that MCoTI-II could exert an effect through binding to the serine protease γ-NGF subunit of the 7S NGF complex, analogous to its binding to its native receptor trypsin. Molecular dynamics simulations showed that MCoTI-II can bind stably to γ-NGF for >350 ns. Modeling indicated that this interaction could sterically inhibit 7S NGF complex formation, potentially altering the equilibrium between inactive complexed and free active NFG protein. In conclusion, the biological study now excludes the MCoTI-II protein as the NGF mimetic factor in the Momordica extract, an important and required step to identify the active component in this seed. On the other hand, the theoretical study has revealed a novel observation that may be of use in the development of strategies to affect NGF activity.