Neurotrophic effects of four commercially important seaweeds and their identified compounds in hippocampal neurons

Abstract

The brain aging is considered as a powerful initiator on the increased susceptibility of neurodegenerative complications, as life expectancy is increasing dramatically. The Alzheimer’s disease, a progressive neurodegenerative disorder, has been implicated with impaired memory even in optimally healthy individuals and found to selectively destroy the central nervous system neurons particularly in the hippocampus. Although, the actual causes of such disease progression remain under explored. The treatment therapies of neurodegenerative disorders are mostly symptomatic, there is no complete cure. Neurotrophic factors in the central nervous system are important for neuronal differentiation, development, outgrowth, function and survival in developing neurons, and have been documented in adult neuritogenesis by the reconstruction of damaged neuronal networks. Therefore, the use of natural compounds or drugs with neurotrophic potential might provide new insights in the treatment of memory related neurological disorders. In an earlier attempt to search potential neurotrophic agents from natural resources that possess neurite outgrowth promoting activity as well as to protect neurons even after injury, I paid my research efforts for screening common seaweeds having neuritogenic effect in primary cultured rat hippocampal neurons. Of the seaweed species tested, an ethanol extract of agar producing seaweed Gracilariopsis chorda (GCE) exhibited potent neurotrophic effect to protect neurons even after injury, followed by Gelidium amansii, Porphyra yezoensis and Undaria pinnatifida. The nerve growth factor stimulating effect of GCE was promoted neuronal cytoarchitecture in developing hippocampal neurons, with an optimal concentration of 30 μg/mL. GCE significantly increased early neuronal differentiation (i.e., polarity and process number) and enhanced axonal and dendritic arborization in a time-responsive manner. Using reverse-phase-high-performance liquid chromatography (RP-HPLC), I quantitated the arachidonic acid (AA) in GCE [0.64% (w/w) of GCE and 1.5 mg/100 g dry weight of G. chorda powder], which significantly accelerated neurite outgrowth similar to GCE. Considering the above findings, I next evaluated the effect of GCE and its active compound AA in promoting dendritic spine dynamics and synaptic plasticity, and their molecular mechanisms in hippcampal neurons at the mature stage in culture. I found that GCE and its AA significantly accelerated the densities and maturation of dendritic filopodia and spines, and eventually synaptogenesis. In western blot and immunocytochemical analyses, treatment of neurons with GCE and AA effectively increased the expression of CDC42 and subsequent ARP2 proteins, which involved in actin organization facilitates the plastic changes of dendritic spines. Functional study provided the similar results that GCE and AA potentiated presynaptic plasticity by increasing the size of synaptic vesicles pool released from axon terminal. Additionally, I investigated whether AA was only the leading compound in GCE that acted like neurotrophic agent. In addition to AA, I detected two other compounds by RP-HPLC analysis which were found to enhance neurite outgrowth in developing neurons, indicating an enormous source of neurotrophic compounds present in GCE. Based on a previously unbiased screening result, the another agar producing seaweed Gelidium amansii has been gaining profound interests due to its robust neurite enhancing abilities in hippocampal neurons. However, no data has been found the neurotrophic effect of G. amansii in adult neuritogenesis using animal model. Therefore, I examined the ethanol extract of G. amansii (GAE) on dendritic morphogenesis and how it promoted structure functional plasticity of dendritic spines associated with spatial learning and memory retention in the hippocampus CA1 neurons to the early age of young mice. The treatment of mice with GAE (0.5 mg/g of body weight; p.o.) significantly increased the dendritic arborization and spine density, and modulated spine dynamics, where a large number of spines were found in mushroom shaped. In immunohistochemical analysis, GAE modulated the expression of synaptic marker proteins and stimulated the co-expression of N-methyl-D-aspartate receptors (NMDARs; NR2A and NR2B) with PSD95, compared to control mice. The active component in GAE was remained to be explored Then, I took the advantages of high-throughput screening for neurotrophic agent from GAE. Thus, I developed a bioassay directed fractionation procedure to identify the active compound from GAE responsible for neurite stimulating effect in cultured rat hippocampal neurons. The results revealed that dichloromethane–ethylacetate (0.2:0.8) fraction partitioned from the chloroform sub-fraction of GAE using open-silica-gel-column was found to increase neurite outgrowth significantly at DIV 2 neurons. For further separation using first RP-HPLC with hexane-ethylacetate solvents as mobile phase, Peak 2 (P2) eluted sample was shown to enhance neurite outgrowth in robust forms. Using second RP-HPLC with ethylacetate-methanol, the P2 sample was further separated into two peaks. Among them, the P2-2 was exhibited as single compound in the RP-HPLC chromatogram that significantly promoted neurites in cultures, indicating a promising source of neurotrophic drug in GAE having a curative measure for neurodegeneration. In a series of previous findings from screening, next, I devoted my further research on popular edible seaweed Porphyra yezoensis to the promotion of neuronal survival and cytoarchitecture and how this alga harmonized the neurotrophic activity to protect neurons during the development. The neurotrophic and neuroprotective activities of the ethanol extract of P. yezoensis (PYE) were investigated in primary cultures of hippocampal neurons. PYE with an optimal concentration of 15 µg/mL significantly increased neurite outgrowth, the number of viable cells, accelerated the rate of neuronal differentiation in cultures, promoted axodendritic arborization, and eventually induced synaptogenesis. In functional analysis, PYE also promoted functional maturation as indicated by the increased expression of presynaptic vesicle pool from axon terminal. Moreover, PYE increased neuronal survivability, which was attributed to reduced level of apoptosis and its ROS scavenging activity. I found taurine was a major organic acid in PYE [23.90 mg/ 100 g dry weight (i.e. 0.024%) of PYE] promoted neurite outgrowth in a dose-dependent manner, and this promotion was suppressed by the taurine antagonist isethionic acid. The results provided evidence that taurine was only the responsible compound in PYE induced neurite outgrowth activity. To extend untiring efforts for searching the most promising neurotrophic substance from seaweed, I performed to investigate more in details the neuritogenic potential of the most widely eaten seaweed Undaria pinnatifida that had shown a promising source of neurite outgrowth promoting substance. Thus, U. pinnatifida could preserve neurons even after the exposure of severe oxidative stress in hippocampal neurons. The death of hippocampal neurons in the CNS is likely to be involved in prolonged and severe oxidative stress. Here, I reported that an ethanol extract of U. pinnatifida (UPE) concentration-dependently increased neuronal viability in both hypoxia-induced oxidative stress and normoxic cultures. UPE, at an optimal concentration of 15 μg/mL, significantly reduced reactive oxygen species formation, DNA fragmentation, early and late apoptosis rates, and mitochondrial membrane dysfunction against hypoxia-induced oxidative shock. In addition, the most active neuroprotectant in UPE was identified as fucoxanthin by RP-HPLC. The amount of fucoxanthin was estimated as 0.32 mg/g of UPE in dry weight, which represents 2.06 mg/100 g of seaweed powder. Bioassay guided isolation of fucoxanthin from UPE dose dependently increased the number of viable neurons with an optimal concentration of 10 μg/mL, and also enhanced the length of neurites. It provided protection from neurite breakage in hypoxia-treated cultures, suggesting the ability to reconstruct the damaged neuronal networks in the CNS.