Heat Shock Cognate 70 kDa Protein Is the Target of Tetradecyl 2,3-Dihydroxybenzoate for Neuritogenic Effect in PC12 Cells

Tetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead compound derived from gentisides with a remarkable neuritogenic activity. However, the target of ABG-001 is yet to be defined to date. In this study, the potential target of ABG-001 was investigated via an activity-based protein profiling (ABPP) analysis, which is a chemical proteomic method for target identification by using chemical probes. Results indicated that the potential target proteins of ABG-001 were heat shock cognate 70 kDa protein (Hsc70), 78 kDa glucose-regulated protein (GRP78), and 14-3-3 theta protein. Then, the potential target of ABG-001 was confirmed by using inhibitors, the cellular thermal shift assay (CETSA) and small-interfering RNA (siRNA) analysis. The inhibitor of Hsc70 and siRNA significantly decreased the neurite outgrowth induced by ABG-001. Furthermore, ABG-001 induced neurite outgrowth was reduced by siRNA against Hsc70, and the results of CETSA suggested that Hsc70 showed a significant thermal stability-shifted effect upon ABG-001 treatment. These results indicated that Hsc70 is the target protein of ABG-001 in PC12 cells.

Neuritogenic Activities of Various Kappaphycus alvarezii Extracts in Hippocampal Neurons

The carrageenophyte Kappaphycus alvarezii seaweed is known to have neuritogenic activities. Post-harvest treatment of seaweed affects its biological activities. Five drying treatments of K. alvarezii, including oven-drying, sun-drying, freeze-drying, shade-drying, and salting followed by shade-drying, were evaluated for their effects on neuritogenic activities. We also evaluated the neuritogenic activity of different parts of K. alvarezii thalli and its carrageenan waste. Neuron cells of the 19th day pregnant rat fetuses were collected from the hippocampus by brain dissection. Neuron cells were isolated by dissociation of the hippocampal tissue. Cells were plated onto poly-DL-lysine-coated glass coverslips in 24-well plates and treated with extracts. All tested extracts were obtained from maceration using 95% ethanol. Freeze and shade-drying extracts exhibited significantly higher neuritogenic activities (p 0.05) compared to that of the vehicle control. Carrageenan waste also significantly promoted the neuritogenic activities (p 0.05) with an optimal dose at 1 µg mL-1. Old and young thalli showed insignificant differences in neuritogenic activities. The carrageenan waste extract retained neuritogenic activities. Thus, the utilization of carrageenan waste for neuritogenic material provides added value to the waste in the carrageenan industry. Freeze and shade dried K. alvarezii can be used as a neuritogenic agent to provide optimum biological activity.

Neuritogenic and Neuroprotective Activities of the Essential Oil From Rhizomes of Curcuma alismatifolia

The essential oil from rhizomes of Curcuma alismatifolia Gagnep. was extracted by hydrodistillation. Chemical analysis by gas chromatography-mass spectrometry identified 12 compounds, of which the most prominent were xanthorrhizol (82.2%), ar-curcumene (6.5%), α-cedrene (1.8%), and β-bisabolol (1.1%). The essential oil at 1 ng/mL showed neuroprotective and neuritogenic activity on P19-derived neurons by significantly enhancing cell viability, length, and branching numbers of the cultured P19-derived neurons. In addition, xanthorrhizol, the major compound of the essential oil, at 10 ng/mL also exhibited the protection of P19-derived neurons. However, the mechanism of their neuroprotective activity was not correlated with their antioxidant activity.

Bioactivity-Guided Isolation of Neuritogenic Factor from the Seeds of the Gac Plant (Momordica cochinchinensis)

Nerve growth factor (NGF) is an endogenously produced protein with the capacity to induce central nervous system (CNS) neuronal differentiation and repair. NGF signaling involves its binding to tropomyosin-related kinase (Trk) receptors, internalization, and initiation of phosphorylation cascades which cause microtubule reorganization and neuronal outgrowth. Because NGF cannot cross the blood-brain barrier, its therapeutic use is limited. Synthetic peptides that can act as NGF receptor agonists (NGF mimetics) are known to attenuate neurodegenerative pathologies in experimental models of Alzheimer’s disease and Parkinson’s disease; however, the existence of plant-based NGF mimetics is uncertain. For this reason, we recently completed a high throughput screening of over 1100 nutraceuticals (vitamins, herbal plant parts, polyphenolics, teas, fruits, and vegetables) to identify neuritogenic factor using a PC-12 cell model. Remarkably we found only one, commonly known as the seed of Gac plant (Momordica cochinchinensis) (MCS). In the current study, we further investigated this seed for its neuritogenic effect using bioactivity-guided chemical separations. The data show no biological neuritogenic activity in any chemical solvent fraction, where activity was exclusive to the crude protein. MSC crude proteins were then separated by 1D electrophoresis, where the active neuritogenic activity was confirmed to have a molecular mass of approximately 17 kDa. Subsequently, the 17kDa band was excised, digested, and run on a UPLC-MS/MS with a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with data evaluated diverse tools such as X! Tandem, OMS, and K-score algorithms. Proteomic evaluation of the 17kDa band confirmed evidence for 11S globulin subunit beta, napin, oleosin, Momordica trypsin inhibitors (TI) MCoTI-I /II, and many isoforms of Two Inhibitor Peptide Topologies (TIPTOPs). While all peptides identified correspond to the genus/species, Momordica cochinchinensis and Cucumis Sativus, a significant limitation of the analysis is the nonexistence of full annotation for the Momordica cochinchinensis proteome. In conclusion, these findings demonstrate that there is a stable protein within MCS having a mass of 17kDa with the capacity to induce neurite outgrowth. Future work will be required to establish the therapeutic value of the MCS for the treatment of neurodegenerative diseases.

Calotropis gigantea Promotes Neuritogenesis and Synaptogenesis through Activation of NGF-TrkA-Erk1/2 Signaling in Rat Hippocampal Neurons

Calotropis gigantea (L.) R. Br (Apocynaceae) (commonly known as milkweed or crown flower) is a large shrub native to temperate regions of Asia, including China, Bangladesh and India and has a long history of use in traditional medicines. In this study, we investigated the neuromodulatory effects of the ethanol extracts of C. gigantea leaves (CGE) during synaptogenesis in the late stage of neuronal development and during early stage neuritogenesis in cultured rat hippocampal neurons. Maximum neuritogenic activity was achieved at a CGE concentration of 7.5[Formula: see text][Formula: see text]g/ml. At this concentration, CGE facilitated the early development of cytoarchitecture, as evidenced by increases in morphometric parameters, such as, the numbers, lengths, and number of branches of initial neurites, axon and dendrites. During the synaptogenic stage (DIV 14), immunocytochemistry (ICC) showed that CGE upregulated synaptic vesicle 2 (SV2, a marker of axon terminals) and postsynaptic density-95 (PSD-95, a postsynaptic marker) and their colocalization. CGE upregulated nerve growth factor (NGF) and activated extracellular signal-regulated kinase 1/2 (Erk1/2), which is blocked by a TrkA-specific inhibitor suggesting the neuritogenic and synaptogenic potential of CGE was due to the activation of NGF-TrkA-Erk1/2 signaling. Moreover, UPLC of CGE did not detect stigmasterol, an active component of C. gigantea. However, the chloroform-methanol and ethyl acetate subfractions of CGE exhibited initial neuritogenic activity, suggesting that multiple active components were responsible for the neurotrophic-mimetic properties of CGE. Our data prove the neuromodulatory ability of CGE and provide a means of identifying new active phytochemicals with potential nootropic, preventative or therapeutic effects on the human brain.