Post-mitotic central nervous system (CNS) neurons have limited capacity for regeneration, creating a challenge in the development of effective therapeutics for spinal cord injury or neurodegenerative diseases. Furthermore, therapeutic use of human neurotrophic agents such as nerve growth factor (NGF) are limited due to hampered transport across the blood brain barrier (BBB) and a large number of peripheral side effects (e.g. neuro-inflammatory pain/tissue degeneration etc.). Therefore, there is a continued need for discovery of small molecule NGF mimetics that can penetrate the BBB and initiate CNS neuronal outgrowth/regeneration. In the current study, we conduct an exploratory high-through-put (HTP) screening of 1144 predominantly natural/herb products (947 natural herbs/plants/spices, 29 polyphenolics and 168 synthetic drugs) for ability to induce neurite outgrowth in PC12 dopaminergic cells grown on rat tail collagen, over 7 days. The data indicate a remarkably rare event-low hit ratio with only 1/1144 tested substances (<111.25 µg/mL) being capable of inducing neurite outgrowth in a dose dependent manner, identified as; Mu Bie Zi, Momordica cochinchinensis seed extract (MCS). To quantify the neurotrophic effects of MCS, 36 images (n = 6) (average of 340 cells per image), were numerically assessed for neurite length, neurite count/cell and min/max neurite length in microns (µm) using Image J software. The data show neurite elongation from 0.07 ± 0.02 µm (controls) to 5.5 ± 0.62 µm (NGF 0.5 μg/mL) and 3.39 ± 0.45 µm (138 μg/mL) in MCS, where the average maximum length per group extended from 3.58 ± 0.42 µm (controls) to 41.93 ± 3.14 µm (NGF) and 40.20 ± 2.72 µm (MCS). Imaging analysis using immunocytochemistry (ICC) confirmed that NGF and MCS had similar influence on 3-D orientation/expression of 160/200 kD neurofilament, tubulin and F-actin. These latent changes were associated with early rise in phosphorylated extracellular signal-regulated kinase (ERK) p-Erk1 (T202/Y204)/p-Erk2 (T185/Y187) at 60 min with mild changes in pAKT peaking at 5 min, and no indication of pMEK involvement. These findings demonstrate a remarkable infrequency of natural products or polyphenolic constituents to exert neurotrophic effects at low concentrations, and elucidate a unique property of MCS extract to do so. Future research will be required to delineate in depth mechanism of action of MCS, constituents responsible and potential for therapeutic application in CNS degenerative disease or injury.
The discovery of nerve growth factor (NGF) mimetics could have the potential application for Alzheimer's disease prevention and therapy. Previously, we have conducted a high‐throughput (HTP) screening of over 1100 nutraceutical products (herbs, vitamins, plant‐based phytochemicals, etc.) to determine if any dietary based substance could induce neurite outgrowth similar to 7S NGF at <200ug/mL in PC‐12 cells. Only a single biologically active with compelling NGF mimetic capacity in the seed of Mu Bie Zi, Momordica cochinchinensis (bitter cucumber) (MCS). The objective of the current study is to identify the bioactive substances within the ground seed pit by testing for NGF biological activity in the crude seed solvent extractions and purified proteins. The active protein fraction was subjected to one‐dimensional gel electrophoresis (native), gel staining, sectioning / excision, electro‐elution back into solution and re‐evaluation for NGF mimetic effects. Results obtained show that the NGF mimetic resided in a low abundant visually absent gel section at around 17 kD. The 17kD gel slice was excised, digested and run on a UPLC‐MS/MS –with a Q Exactive Hybrid Quadrupole ‐ Orbitrap Mass Spectrometer. The data were evaluated using X! Tandem, OMS, X Hunter and K‐score algorithms. Proteomic evaluation of the 17kD band was conducted showing the presence of 11S globulin subunit beta, napin, oleosin 18.2 kDa, trypsin inhibitor 1 MCoTI‐I /II (known to contain growth factor cystine knots) and Two Inhibitor Peptide Topologies TIPTOPs (isoforms 1,2,3,5,6), all with taxonomical identities inherent to the cucumber family (Momordica cochinchinensis, macrophylla, charantia, Cucumis sativus).Support or Funding InformationThis research was supported by the National Institute for Minority Health and Health Disparities of the National Institutes of Health through Grant Number G12 MD007582 and Grant Number P20 MD006738
The capacity of the human brain to enable growth of neurons occurs through neural stem cell differentiation guided by trophic factors such as brain‐derived neurotrophic factor or nerve growth factor (NGF). Elevated levels of NGF are believed to play a protective role in preventing a diverse range of age related neurodegenerative conditions. Moreover, PC‐12 dopaminergic cells are a valuable model for evaluating neuroprotective / trophic agents relevant to Parkinson's disease. In this study, we screened 1145 compounds [29 flavonoids, 168 synthetic drugs, 948 herb, fruit and vegetable extracts] for NGF mimetic effects in PC‐12 cells in the absence of NGF. The data indicate that 2 natural products of the 1145 tested at sub‐lethal concentrations had capacity to initiate neurite outgrowth comparable to NGF, which acquired properties of sympathetic neurons. Differentiation was analyzed by fluorescent immunocytochemistry Alexa (488)‐conjugated phalloidin / propidium iodide nuclear counterstain and contrast microscopy in live cells. These data support very unique properties of few natural products to mimic the effects of NGF. Grant Funding Source: Supported by NIMHD G12 MD007582 and P20 MD006738
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