Proteomic Analysis of the Neurotrophic Effect of Gelidium amansii in Primary Cultured Neurons

Abstract

Gelidium amansii is an edible and economically important red alga consumed in South Eastern Asia. In previous studies, we reported that the ethanol extracts of G. amansii (GAE) has promising modulatory activity with respect to the morphological and functional maturation of hippocampal neurons in culture. In this study, we show that the chloroform (CHCl₃) subfraction of GAE and the ethyl acetate (EtOAc) fraction dose-dependently promoted neurite outgrowth, and their effects were comparable with that of GAE. We further assessed in cultured cortical neurons, proteins differentially expressed in the presence/absence of the GAE, CHCl₃ subfraction, and the EtOAc fraction by 2D-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomic data revealed that a number of proteins responsible for multiple cellular and biochemical functions vital for neuronal development and maturation were significantly upregulated in neurons treated with the GAE, CHCl₃ subfraction, and the EtOAc fraction. Of the identified proteins, profilin 2a, septin 7, cdc42, protein phosphatase 2A, DA11, eukaryotic translation initiation factor 5A-1, and γ-enolase are known to play important roles in neuritogenesis and dendritic arborization. Immunofluorescence data demonstrate that GAE-treated hippocampal neurons showed greater intensity ratios in the expressions of the septin 7 and cdc42 compared to vehicle control, validating their proteomic profiles. Together these results suggest that the GAE/CHCl₃ subfraction and EtOAc fraction promote neurite development by up or downregulating several key proteins.

Introduction

Physiologically, neurons are supported by a number of trophic factors, from the embryonic stage through life. These neurotrophic factors (NFs) regulate the expressions of multiple proteins essential for the normal functioning of neurons. Accordingly, insufficiencies of trophic supports might be responsible for neuronal dysfunctions and the subsequent developments of neurological disorders like Alzheimer's disease.^1,2 Since endogenous NFs are no longer sufficient to support normal brain activity in the aging and degenerating brain, the introduction of exogenous neurotropic factors represents a treatment strategy.

Dendritic atrophy with synapse and spine loss is observed in the aging human brain.³ Reduction in dendritic arbor size is also associated with psychiatric illnesses, such as schizophrenia and major depressive disorder, as well as neurodegenerative diseases, such as Alzheimer's disease.^4,5 Neurotrophins have proven to promote neuritogenesis, synaptogenesis, and neuronal survival.^6
–8 The brain-derived neurotrophic factor (BDNF) promotes neurogenesis.^9,10 BDNF gene transfection triggers dendritic and axonal branching in dentate gyrus granule cell cultures.¹¹ Therefore, there are strong rationales suggesting that increasing supply of the neurotrophins to degenerating neurons may be a potent way to restore neuronal function in neurodegenerative conditions.

Identification of neuritogenic and neurotrophic molecules from natural sources may provide important drug leads for activation of neuroregeneration in various forms of neuronal injuries. Macroalgae have been explored as potential sources of bioactive compounds and have been reported to possess various pharmacological effects, including antioxidative, antiaging, anticancer,¹² and antiobesity¹³ activities. Furthermore, several authors have demonstrated the suitability of marine algae for neurotrophic research.^14
–16 In addition, recently, we reported the neurotrophic activities of marine algae, particularly Gelidium amansii, Sargassum fulvellum, and Undaria pinnatifida, in primary cultures of hippocampal neurons.^17
–19

In this study, we performed proteomic analysis to identify molecular target proteins up- or downregulated during neuronal development in vitro. We demonstrate that the ethanol extracts of G. amansii (GAE), CHCl₃ subfraction, and EtOAc fraction can promote neurite outgrowth by regulating the expressions of proteins responsible for neurite synthesis and maturation.

Materials and Methods

Extraction and fractionation of G. amansii

The ethanol extract of G. amansii was prepared as described previously.¹⁸ Briefly, a pulverized algal sample was extracted with 95% aqueous ethanol and filtered through sterile cotton. The filtrate was concentrated in vacuo and completely dried to produce GAE. The filtration residue was re-extracted with EtOAc to obtain the EtOAc fraction. A flowchart of solvent fractionation is provided in Figure 1 (modified).²⁰ GAE, its subfractions, and the EtOAc fraction were dissolved in dimethyl sulfoxide (DMSO) at 16 mg/mL and stored in foil-wrapped vials at −20°C in the dark before experiments.

FIG. 1.

Flowchart of the extraction and fractionation procedure.

Primary neuronal culture and treatment

Animal care and use were in accord with the institutional guidelines issued by the Institutional Animal Care and Use Committee of the Dongguk University College of Medicine and were approved by the same committee. Rat (Sprague-Dawley) primary neuronal cultures (dissociated hippocampal cells for immunocytochemistry and dissociated cortical cells for proteomics) were prepared as previously described^18,21 with the following exceptions: dissociated cells were seeded onto poly-dl-lysine (Sigma-Aldrich, St. Louis, MO, USA) coated Ø12-mm glass coverslips in 24-well culture plates at a density of 1.0 × 10⁴ cells/cm² for the morphological study or at 1.0 × 10⁶ cells/cm² for proteomic analysis. Cultures were maintained in a defined serum-free Neurobasal media supplemented with B27 and incubated at 37°C under a 5% CO₂/95% air atmosphere. Sample extracts or vehicle (DMSO, final concentration 0.1% [v/v]) was added to culture media before cell seeding. For neurite outgrowth promoting activity, GAE, its subfractions, and the EtOAc fraction were added at 3.75 to 30 μg/mL. For proteomic and immunocytochemical analyses, GAE, CHCl₃ subfraction, and EtOAc fraction were used at 15, 7.5, and 15 μg/mL, respectively.

Fluorescence labeling with DiO

At DIV3 (3 days in vitro), neurons were live-stained with Vybrant DiO (Molecular Probes, Eugene, OR, USA), according to the manufacturer's instructions. DiO is a lipophilic dye that binds to plasma membranes, and thus, enables entire neurons to be visualized.

Image acquisition and analysis

A Leica Research Microscope (DM IRE2) equipped with I3 S, N2.1S, and Y5 filter systems (Leica Microsystems AG, Wetzlar, Germany) was used for phase-contrast and epifluorescence microscopy. Images (1388 × 1039 pixels) were acquired using a high-resolution CoolSNAP™ CCD camera (Photometrics, Tucson, AZ, USA) using Leica FW4000 software. Morphometric analyses and quantification were performed using ImageJ (version 1.45) software with the simple neurite tracer plug-in (National Institute of Health, Bethesda, MD, USA). Fluorescent images taken of DiO stained cultures were converted into grayscale mode and inverted. Morphometric parameters, such as the numbers of primary neurites (neurites that originated directly from soma), total primary neurite lengths (the sum of the lengths of primary neurites), and the numbers of branching points, were measured.

Extraction of protein sample

At DIV6, proteins were harvested from cultured neurons using the Nuclei EZ Prep Nuclei Isolation Kit (Sigma-Aldrich). Protein concentrations were determined using the Bradford method.²²

2D-PAGE and image analysis

2D-PAGE was performed on whole-cell lysate protein fractions. IPG dry strips (4–10 NL IPG, 24 cm; Genomine, Inc., Pohang, Korea) were equilibrated for 12–16 h in a 7 M urea, 2 M thiourea solution containing 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% Pharmalyte and loaded with 200 μg of samples. Isoelectric focusing (IEF) was performed at 20°C using a Multiphor II electrophoresis unit and EPS 3500 XL power supply (GE Healthcare, Little Chalfont, UK), according to the manufacturer's instructions. For IEF, the voltage was linearly increased from 150 to 3500 V over 3 h for sample entry followed by a constant 3500 V. Complete focusing was achieved at after 96 kVh. Before the second dimension, strips were incubated for 10 min in equilibration buffer (50 mM Tris-Cl, pH 6.8 containing 6 M urea, 2% SDS, and 30% glycerol), first with 1% DTT and second with 2.5% iodoacetamide. Equilibrated strips were inserted into SDS-PAGE gels (20 × 24 cm, 10–16%). SDS-PAGE was performed using the Hoefer DALT 2D system (Amersham Biosciences), according to the manufacturer's instructions. Two-dimensional gels were run at 20°C for 1700 Vh and then silver stained as described.²³ Quantitative analysis of digitized images was carried out using PDQuest software (version 7.0, Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. Spot intensities were normalized versus total valid spot intensities. Protein expressions over or under 1.5-fold versus the vehicle control were further analyzed.

Protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting

For protein identification by peptide mass fingerprinting (PMF), protein spots were excised, digested with trypsin (Promega, Madison, WI, USA), mixed with α cyano-4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% TFA, and subjected to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis (Microflex LRF 20; Bruker Daltonics, Billerica, MA, USA), as described.²⁴ Spectra were collected using 300 shots per spectrum over the m/z range 600–3000 and calibrated by two-point internal calibration using trypsin autodigestion peaks (m/z 842.5099 and 2211.1046). Peak lists were generated using Flex Analysis version 3.0. The thresholds used for peak-picking were as follows: 500 for minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT, developed by The Matrix Science (www.matrixscience.com/), was used for protein identification by PMF. The following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of ±0.1 Da. PMF protein identification was accepted by probability-based scoring.

Immunocytochemistry

Neurons on coverslips were rinsed briefly with Dulbecco's phosphate-buffered saline (D-PBS; Invitrogen, Carlsbad, CA, USA) and fixed by a sequential paraformaldehyde/methanol fixation procedure²⁵ on DIV12. For immunostaining, the following antibodies were used: primary antibodies to tubulin α-subunit (mouse monoclonal 12G10, 1:1000 dilution; Developmental Studies Hybridoma Bank, University of Iowa, USA); Septin 7 (rabbit polyclonal, 1:500 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); and Cdc42 (rabbit polyclonal, 1:200 dilution; Thermo Fisher Scientific, Waltham, MA, USA) and secondary antibodies (Alexa Fluor 488-conjugated goat anti-mouse IgG [1:1000] and Alexa Fluor 568-conjugated donkey anti-rabbit IgG [1:1000], Molecular Probes). Fixed neurons were incubated with primary antibody followed by secondary antibody and mounted on slides as described.²⁵ Fluorescent images (1388 × 1039 pixels) were acquired as described in an earlier section. Puncta for Septin 7, Cdc42, and α-tubulin were counted per 50 μm length of dendrite using puncta analyzer plug-in.²⁶ The puncta intensity ratios for Septin 7/α-tubulin and Cdc42/α-tubulin were then measured.

Statistical analysis

Results are expressed as the means ± standard error of means (SEMs) of at least three independent experiments. Statistical comparisons were made using one-way analysis of variance (ANOVA) with Duncan's post hoc multiple comparisons using SPSS ver. 16.0. Statistical significance was accepted for P values <.05.

Results

Neurite outgrowth promoting activities of GAE, its subfractions, and of the EtOAc fraction

In a previous report, we showed that GAE has promising neuritogenic activity in primary neuronal cultures.¹⁸ In this study, we investigated whether GAE subfractions and the EtOAc fraction have similar neuritogenic activities. Hippocampal neurons were incubated with various concentrations of GAE or its subfractions (CHCl₃, methanol [MeOH], CHCl₃-MeOH, or aqueous subfractions) or the EtOAc fraction (Fig. 1). In this study, we only present the data of fractions that exhibited visible neuritogenic activity. Typical microscopic fields of cultures grown with GAE, or its CHCl₃ subfraction, or EtOAc fraction at 3.75 to 30 μg/mL for 3 days are shown in Figure 2A. Both the EtOAc fraction and the CHCl₃ subfraction significantly and dose-dependently increased neurite numbers, lengths, and branchings. Furthermore, their effects were comparable with that of GAE (Fig. 2B). Notably, at 15 and 30 μg/mL, the CHCl₃ subfraction had little inhibitory effect, but at lower concentrations it had neuritogenic effects.

FIG. 2.

Dose-dependent neuritogenic activities of GAE, the CHCl₃ subfraction, and of the EtOAc fraction in embryonic (E19) rat hippocampal neurons cultured on poly-dl-lysine-coated coverslips in vehicle (DMSO, <0.5% [v/v]), GAE, the CHCl₃ subfraction, or the EtOAc fraction at the indicated concentrations for 3 days; neurons were live-stained with DiO. (A) Representative photomicrographs. Scale bar = 80 μm in all images. (B) Morphometric analysis of branching points (a), number (b) and total lengths of primary neurites (TLPN) (c) demonstrated that extracts promoted neurite outgrowth in a dose-dependent manner. Statistically significant versus vehicle controls. *P < .05, **P < .01, and ***P < .001 (ANOVA). ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; GAE, ethanol extracts of Gelidium amansii.

Proteomic profiles of cortical neurons treated with GAE, its subfractions, or the EtOAc fraction

2-DE based proteomic analysis was carried out on cortical neurons treated with GAE, the CHCl₃ subfraction, the EtOAc fraction, or vehicle to evaluate changes in protein expression profiles. Based on their neurite outgrowth promoting activity, we used GAE and EtOAc fraction at 15 μg/mL and CHCl₃ subfraction at 7.5 μg/mL for proteomic analysis. Because large amounts of sample are needed for proteomic analysis, cortical neurons were used instead of hippocampal neurons. In primary neuronal cultures, the neurite promoting activity of extracts commenced as soon as undifferentiated neurons attached to surfaces. Although dendritic arbor formation started after 4 days in culture, the molecular events associated with dendritic arborization were induced between day 5 and 6 in culture. Thus, 6 days of incubation was chosen for proteomic analysis. Representative 2D-PAGE images are shown in Figure 3. More than 800 protein spots were detected in each gel, and 200 spots were differentially expressed. Among them, 14 showed significant upregulation and 4 significant downregulation (indicated by circles in Fig. 3). Figure 4 shows magnified spot images of the identified proteins and the relative intensities of each protein spot.

FIG. 3.

Representative 2-DE gel images. Embryonic (E19) rat cortical neurons were cultured on poly-dl-lysine-coated coverslips with vehicle (DMSO, <0.5% [v/v]) (A), GAE (B), the CHCl₃ subfraction (C), or the EtOAc fraction (D) at the indicated concentrations for 6 days. Proteins were harvested and identified as described in Materials and Methods section. Differentially expressed proteins are indicated by circles and labeled with numbers, which correspond to those shown in Table 1. Thick circles indicate downregulated proteins. The pI range is below the gels, and molecular mass is indicated on the right.

FIG. 4.

Intensities of differentially expressed proteins. (A) Magnified images of protein spots up- (a) or downregulated (b) in cortical neurons by GAE, the CHCl₃ subfraction, or the EtOAc fraction. (B) Relative spot intensities were calculated as ratios versus vehicle-treated controls. Asterisks indicate downregulated spots.

Identification of differentially expressed protein spots by MS

A total of 17 proteins were unequivocally identified by MALDI-TOF-MS mass spectrometry (MALDI-TOF-MS) and PMF. The characteristics of the identified proteins are summarized in Table 1.

Table 1.

Summary of Differentially Expressed (up/down) Proteins in Rat Embryonic Cortical Neurons in Culture Treated with GAE, the CHCl₃ Subfraction, or the EtOAc Fraction

						Spot intensity (fold change over control)
Spot no.	Gi-accession no.	Identified protein name	Abbreviated protein name	Mascot score	Mass (kD)/pI	GAE	CHCl₃subfraction	EtOAc fraction	Cellular/biochemical functions
604	202549	Iodothyronine 5′ monodeiodinase	ITMD	143	54.4/4.87	2.6	1.1	2.0	Metabolism
702	8394027	Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A	PP2A	125	66.1/5.00	2.0	1.0	1.5	Metabolism, the cell cycle, cell proliferation, replication, transcription, and translation
703	8394027	Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A	PP2A	119	66.1/5.00	12.0	3.0	4.7	Metabolism, the cell cycle, cell proliferation, replication, transcription, and translation
704	71534276	Rab GDP dissociation inhibitor alpha	RabGDIα	165	51.1/5.00	2.2	1.7	2.1	Intracellular membrane sorting and membrane trafficking
1003	4503545	Eukaryotic translation initiation factor 5A-1	eIF5A	112	17.1/5.08	4.5	9.9	2.6	Protein synthesis
1401	26023949	Gamma-enolase	γ-enolase	183	47.5/5.03	1.6	3.2	1.5	Glycolysis
3811	209862801	Alanine-tRNA ligase, cytoplasmic	AtRL	182	107.5/5.41	−0.2	−0.8	−0.2	Alanine and aspartate metabolism and aminoacyl-tRNA biosynthesis
4813	126723582	Thimet oligopeptidase	TOP	327	79.2/5.64	−0.5	−0.1	−0.4	Proteolytic enzyme, able to generate delta-opioid agonists like Met- and Leu-enkephalin
5007	223365703	Chain A, crystal structure of rat Profilin 2a	PFN2a	92	15.5/6.75	2.2	1.6	1.4	Cytoskeleton/G-actin binding protein
5008	16357472	Cell division control protein 42 homolog	Cdc42	97	21.7/5.76	1.7	1.4	1.5	Cell division/GTPase activating protein
5010	16758810	Ubiquitin-conjugating enzyme E2N	UC-E2N	102	17.2/6.13	3.6	2.3	2.1	Ubiquitination
5106	149030731	Proteasome subunit, beta type 4	PS-β4	94	29.5/5.65	2.0	1.3	1.8	Protein degradation
5812	81889021	Glycine-tRNA ligase	GtRL	82	72.7/5.76	−0.2	−0.4	−0.2	Aminoacyl-tRNA biosynthesis
6207	8394060	Proteasome subunit alpha type-1	PS-α1	107	29.8/6.15	7.6	5.7	6.1	Protein degradation
6819	62945278	2-Oxoglutarate dehydrogenase, mitochondrial precursor	2-OGD	68	117.4/6.3	−0.0	−0.1	−0.0	Energy production and synthesis of neurotransmitter, glutamate
7008	1836058	DA11		90	15.3/6.14	5.9	1.0	1.0	Fatty acid binding protein
7106	16758470	Platelet-activating factor acetylhydrolase IB subunit gamma	PAF-AH	159	25.9/6.42	2.0	2.7	2.1	Platelet-activating factor catabolism
8111	149027897	Septin 7, isoform CRA_b	Sept7	65	51.7/8.51	1.0	2109	555.0	Cytoskeleton

GAE, ethanol extracts of Gelidium amansii.

Validation of proteomic data by immunocytochemistry

We then performed immunocytochemistry of some representative upregulated proteins such as Septin 7 and Cdc42 to validate their proteomic results using hippocampal neuronal cultures, because they are more homogeneous than cortical ones. Septin 7 is involved in cytoskeleton formation, and Cdc42 is involved in the formation of filopodia and neurite outgrowth. In this study, as in proteomic analysis, we used GAE and EtOAc fraction at 15 μg/mL and CHCl₃ subfraction at 7.5 μg/mL for immunocytochemical analysis. The expressions of septin 7 and cdc42, as determined by the ratios of the fluorescence intensities of septin 7 to α-tubulin and cdc42 to α-tubulin, were significantly higher in the CHCl₃ subfraction and the EtOAc fraction-treated hippocampal neurons than vehicle controls (Fig. 5), suggesting their potentiating roles not only in earlier stages of maturation but also to advanced stages of development. However, GAE itself did not induce the expression of septin 7, but significantly promoted cdc42 expression.

FIG. 5.

Immunocytochemical validation of the expression of some proteomics-identified proteins. Hippocampal neurons were cultured on poly-dl-lysine coated coverslips with GAE or the CHCl₃ subfraction, or the EtOAc fraction or vehicle, for 12 days. Neurons were then fixed and double immunostained for Septin 7/α-tubulin and Cdc42/α-tubulin. (A) Representative fluorescence photomicrographs of Septin 7 versus α-tubulin and Cdc42 versus α-tubulin immunostained hippocampal neurons from vehicle (DMSO, <0.5% [v/v]), GAE, the CHCl₃ subfraction, or the EtOAc fraction–treated culture. Scale bar, 4 μm in all images. (B) Fluorescence intensity ratios of Septin 7 versus α-tubulin (a) and Cdc42 versus α-tubulin (b). Statistically significant versus vehicle controls. *P < .05, **P < .01, and ***P < .001 (ANOVA). Bars represent the mean ± SEM (n = 7–10 individual neurons). SEM, standard error of mean.

Discussion

The expressions of multiple proteins within neurons are attributable to the effects of NFs. Several authors have described the NF-regulated expressions of proteins vital for neuronal differentiation, maturation, and survival.^27,28 In a previous study, we demonstrated that GAE (the filtrate fraction of G. amansii ethanol extracts) has a dose-dependent neurotrophic effect in primary hippocampal neurons.¹⁸ In the present study, we further fractionated G. amansii into different subfractions, and we found that the GAE, its CHCl₃ subfraction, and the EtOAc fraction had neurotrophic activities comparable with that of GAE, whereas the CHCl₃-MeOH, MeOH, and aqueous subfractions of GAE did not exhibit any neurotrophic activity. These findings suggest that GAE contains NF-like compounds that are predominantly extracted by a nonpolar solvent CHCl₃, which suggests they are phenolics.²⁰ The CHCl₃ subfraction showed maximum neuritogenic activity at 7.5 μg/mL concentration, but higher concentrations, especially 30 μg/mL, significantly reduced the activity to the control level. This might be due to the presence of some negative ingredients within CHCl₃ subfraction.

We performed proteomic analysis to explore changes in protein expressions in cortical neurons in response to the presence or absence of the GAE, CHCl₃ subfraction, or its EtOAc fraction. As a result, 18 differentially expressed proteins were identified by 2D-PAGE and MALDI-TOF-MS.

Proteins involved in dendritic arborization

Profilin 2a (spot 5007)

Profilins are key regulators of actin dynamics and thus play roles in the actin-dependent regulation of neuronal architecture. Among the different isoforms, profilin 2a (PFN2a) was upregulated in extract-treated neurons compared with control neurons. PFN2a deficient neurons display significantly less dendritic complexity and fewer spine numbers.²⁹ In another report, it was suggested that profilins have postsynaptic functions with respect to dendritic spine stabilization and synaptic plasticity.³⁰ Thus, it appears that profilin 2a may be involved in dendritic outgrowth in extract-treated neurons.

Septin 7 (spot 8111)

Septin 7 (Sept7) was also highly upregulated in neurons treated with the EtOAc fraction or the CHCl₃ subfraction. Septins are involved in cytokinesis, membrane trafficking, and structural scaffolding and to do so localize at the bases of dendritic protrusions and dendritic branch points.^31
–33 Furthermore, the overexpression of Sept7 increases dendrite branching and the densities of dendritic protrusions, whereas knockdown of Sept7 by RNA interference reduces dendrite arborization. Thus, it seems that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.^31,32 However, why the CHCl₃ subfraction but not GAE itself upregulated Sept7 expression is inexplicable at present.

Proteins involved in neurite outgrowth

Cdc42 (spot 5008)

Many cellular processes that lead to changes in neuronal morphology are under the control of different small GTPases,^25,34 for example, Cdc42 is involved in the formation of filopodia and neurite outgrowth. In the present study, Cdc42 (spot 5008) was upregulated in CHCl₃ subfraction and EtOAc fraction–treated neurons, which suggest that the neurotrophic activities of these fractions are mediated through the upregulation of Cdc42.

Protein phosphatase 2A (spot 702 and 703)

Protein phosphatase 2A (PP2A) is an important serine/threonine protein phosphatase and has been suggested to be crucial for neural development and normal function of the eukaryotic nervous system.³⁵ Liu et al. ³⁶ reported that the upregulation of PP2A promotes extensive outgrowth of long neurites in Neura2A cells and the developments of long single axons or multiple axons in hippocampal neurons. Furthermore, zinc-induced tau hyperphosphorylation, which is responsible for neurofibrillary tangle formation in Alzheimer's disease, was completely reversed by the pharmaceutical and genetic upregulation of PP2A.³⁷ In the present study, PP2A was upregulated several fold when neurons were treated with the CHCl₃ subfraction or the EtOAc fraction, which suggests that they may have therapeutic value during the early phase of AD.

DA11 (spot 7008)

DA11 is a fatty acid binding protein, and the strong association between DA11 gene expression and CNS development suggests that DA11 plays important roles in axonal growth and neuronal differentiation in many different neuronal populations.³⁸ These previous findings indicate that the upregulation of DA11 (spot 7008) by GAE is related to the neurotrophic activity of GAE.

Eukaryotic translation initiation factor 5A-1 (eIF5A; spot 1003)

eIF5A is an important translation machinery protein that is responsible for neuronal survival and neurite extension in the nervous system³⁹ and was found to be upregulated by GAE, the CHCl₃ subfraction, and the EtOAc fraction. eIF5A, the only known protein containing the polyamine-derived amino acid hypusine, mediates neurotrophic and neuroprotective actions of nerve growth factor in neurite outgrowth and cell survival of PC12 cells.⁴⁰

γ-enolase (spot 1401)

γ-enolase was upregulated in CHCl₃ subfraction and EtOAc fraction–treated neurons. γ-enolase is a highly conserved cytoplasmic glycolytic enzyme involved in cell differentiation and growth,^37,41 and neuroprotective and neurite outgrowth effects have been reported for the C-terminal part of γ-enolase.^42,43

Other proteins

Other upregulated proteins include a number of proteins involved in neurotransmission, such as Rab GDP dissociation inhibitor alpha (RabGDIα, spot 704), proteins involved in protein degradation such as ubiquitin-conjugating enzyme E2N (UC-E2N and spot 5010), proteasome subunit, beta type 4 (PS-β4 and spot 5106), and proteasome subunit alpha type-1 (PS-α1 and spot 6207), and proteins in other metabolic pathways such as iodothyronine 5′ monodeiodinase (spot 604) and platelet-activating factor acetylhydrolases (spot 7106). In contrast, a mitochondrial enzyme 2-oxoglutarate dehydrogenase (2-OGD and spot 6819), a neuropeptide-metabolizing enzyme thimet oligopeptidase (spot 4813),⁴⁴ and protein biosynthesis enzymes alanine-tRNA ligase (spot 3811) and glycine-tRNA ligase (spot 5812) were downregulated.

We believe up- or downregulations were caused by the net effects of multiple ingredients in each of these extracts. Further functional studies are required to elucidate the mechanisms by which these proteins are involved in the neurotrophic effects of these extracts.

Footnotes

Acknowledgments

This research was partially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning to ISM (2015R1A2A2A01007104). M.A.H. wishes to thank the Korean National Institute of International Education (NIIED) for graduate assistance.

Author Disclosure Statement

No competing financial interests exist.

References

Siegel

, Chauhan

: Neurotrophic factors in Alzheimer's and Parkinson's disease brain. Brain Res Brain Res Rev, 2000; 33:199–227.

Schaeffer

, Novaes

, da Silva

, Skaf

, Mendes-Neto

: Strategies to promote differentiation of newborn neurons into mature functional cells in Alzheimer brain. Prog Neuropsychopharmacol Biol Psychiatry, 2009; 33:1087–1102.

Dickstein

, Weaver

, Luebke

, Hof

: Dendritic spine changes associated with normal aging. Neuroscience, 2013; 251:21–32.

Anderton

, Callahan

, Coleman

, et al.: Dendritic changes in Alzheimer's disease and factors that may underlie these changes. Prog Neurobiol, 1998; 55:595–609.

Broadbelt

, Byne

, Jones

: Evidence for a decrease in basilar dendrites of pyramidal cells in schizophrenic medial prefrontal cortex. Schizophr Res, 2002; 58:75–81.

Kromer

: Nerve growth factor treatment after brain injury prevents neuronal death. Science, 1987; 235:214–216.

McAllister

, Katz

, Lo

: Neurotrophins and synaptic plasticity. Annu Rev Neurosci, 1999; 22:295–318.

Patapoutian

, Reichardt

: Trk receptors: Mediators of neurotrophin action. Curr Opin Neurobiol, 2001; 11:272–280.

Pencea

, Bingaman

, Wiegand

, Luskin

: Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci, 2001; 21:6706–6717.

10.

Scharfman

, Goodman

, Macleod

, Phani

, Antonelli

, Croll

: Increased neurogenesis and the ectopic granule cells after intrahippocampal BDNF infusion in adult rats. Exp Neurol, 2005; 192:348–356.

11.

Danzer

, Crooks

, Lo

, McNamara

: Increased expression of brain-derived neurotrophic factor induces formation of basal dendrites and axonal branching in dentate granule cells in hippocampal explant cultures. J Neurosci, 2002; 22:9754–9763.

12.

Smit

: Medicinal and pharmaceutical uses of seaweed natural products: A review. J Appl Phycol, 2004; 16:245–262.

13.

Maeda

, Hosokawa

, Sashima

, Funayama

, Miyashita

: Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochem Biophys Res Commun, 2005; 332:392–397.

14.

Ina

, Hayashi

, Nozaki

, Kamei

: Pheophytin a, a low molecular weight compound found in the marine brown alga Sargassum fulvellum, promotes the differentiation of PC12 cells. Int J Dev Neurosci, 2007; 25:63–68.

15.

Tsang

, Kamei

: Sargaquinoic acid supports the survival of neuronal PC12D cells in a nerve growth factor-independent manner. Eur J Pharmacol, 2004; 488:11–18.

16.

Tsang

, Ina

, Goto

, Kamei

: Sargachromenol, a novel nerve growth factor-potentiating substance isolated from Sargassum macrocarpum, promotes neurite outgrowth and survival via distinct signaling pathways in PC12D cells. Neuroscience, 2005; 132:633–643.

17.

Hannan

, Kang

, Hong

, et al.: A brown alga Sargassum fulvellum facilitates neuronal maturation and synaptogenesis. In Vitro Cell Dev Biol Anim, 2012; 48:535–544.

18.

Hannan

, Kang

, Hong

, et al.: The marine alga Gelidium amansii promotes the development and complexity of neuronal cytoarchitecture. Phytother Res, 2013; 27:21–29.

19.

Hannan

, Mohibbullah

, Hong

, et al.: Gelidium amansii promotes dendritic spine morphology and synaptogenesis, and modulates NMDA receptor-mediated postsynaptic current. In Vitro Cell Dev Biol Anim, 2014; 50:445–452.

20.

Harborne

: Phytochemical Methods: A Guide to Modern Technique of Plant Analysis. Chapman & Hall, London, 1984.

21.

Sharif

, Islam

, Moon

: N-acetyl-D-glucosamine kinase interacts with dynein-Lis1-NudE1 complex and regulates cell division. Mol Cells, 2016; 39:669–679.

22.

Bradford

: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976; 72:248–254.

23.

Oakley

, Kirsch

, Morris

: A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem, 1980; 105:361–363.

24.

Fernandez

, Gharahdaghi

, Mische

: Routine identification of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Electrophoresis, 1998; 19:1036–1045.

25.

Moon

, Cho

, Jin

, Walikonis

: A simple method for combined fluorescence in situ hybridization and immunocytochemistry. Mol Cells, 2007; 24:76–82.

26.

Ippolito

, Eroglu

: Quantifying synapses: An immunocytochemistry-based assay to quantify synapse number. J Vis Exp, 2010; 45:e2270.

27.

Liao

, Pilotte

, Xu

, et al.: BDNF induces widespread changes in synaptic protein content and up-regulates components of the translation machinery: An analysis using high-throughput proteomics. J Proteome Res, 2007; 6:1059–1071.

28.

Manadas

, Santos

, Szabadfi

, et al.: BDNF-induced changes in the expression of the translation machinery in hippocampal neurons: Protein levels and dendritic mRNA. J Proteome Res, 2009; 8:4536–4552.

29.

Murk

, Wittenmayer

, Michaelsen-Preusse

, et al.: Neuronal profilin isoforms are addressed by different signaling pathways. PLoS One, 2012; 7:e34167.

30.

Ackermann

, Matus

: Activity-induced targeting of profilin and stabilization of dendritic spine morphology. Nat Neurosci, 2003; 6:1194–1200.

31.

Tada

, Simonetta

, Batterton

, Kinoshita

, Edbauer

, Sheng

: Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons. Curr Biol, 2007; 17:1752–1758.

32.

Xie

, Vessey

, Konecna

, Dahm

, Macchi

, Kiebler

: The GTP-binding protein Septin 7 is critical for dendrite branching and dendritic-spine morphology. Curr Biol, 2007; 17:1746–1751.

33.

Cho

, Lee

, Dutta

, Song

, Walikonis

, Moon

: Septin 6 regulates the cytoarchitecture of neurons through localization at dendritic branch points and bases of protrusions. Mol Cells, 2011; 32:89–98.

34.

Nobes

, Hall

: Rho, rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell, 1995; 81:53–62.

35.

Janssens

, Goris

: Protein phosphatase 2A: A highly regulated family of serine/threonine phosphatases implicated in cell growth and signaling. Biochem J, 2001; 353:417–439.

36.

Liu

, Zheng

, Luo

, Wang

, Zhu

: Effect of PP-2A on neurite outgrowth in neuronal cells. In Vitro Cell Dev Biol Anim, 2010; 46:702–707.

37.

Xiong

, Jing

, Zhou

, et al.: Zinc induces protein phosphatase 2A inactivation and tau hyperphosphorylation through Src dependent PP2A (tyrosine 307) phosphorylation. Neurobiol Aging, 2012; 34:745–756.

38.

Liu

, Molina

, Welcher

, Longo

, De León

: Expression of DA11, a neuronal-injury-induced fatty acid binding protein, coincides with axon growth and neuronal differentiation during central nervous system development. J Neurosci Res, 1997; 48:551–562.

39.

Willis

, Twiss

: The evolving roles of axonally synthesized proteins in regeneration. Curr Opin Neurobiol, 2006; 16:111–118.

40.

Huang

, Higginson

, Hester

, Park

, Snyder

: Neuronal growth and survival mediated by eIF5A, a polyamine-modified translation initiation factor. Proc Natl Acad Sci USA, 2007; 104:4194–4199.

41.

Schmechel

, Brightman

, Marangos

: Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation. Brain Res, 1980; 190:195–214.

42.

Hattori

, Takei

, Mizuno

, Kato

, Kohsaka

: Neurotrophic and neuroprotective effects of neuron-specific enolase on cultured neurons from embryonic rat brain. Neurosci Res, 1995; 21:191–198.

43.

Hafner

, Obermajer

, Kos

: γ-Enolase C-terminal peptide promotes cell survival and neurite outgrowth by activation of the PI3K/Akt and MAPK/ERK signalling pathways. Biochem J, 2012; 443:439–450.

44.

Yamanaka

, Lebrethon

, Vandersmissen

, et al.: Early prepubertal ontogeny of pulsatile gonadotropin-releasing hormone (GnRH) secretion. I. Inhibitory autofeedback control through prolyl endopeptidase degradation of GnRH. Endocrinology, 1999; 140:4609–4615.