Animals

Embryos (at the embryonic day of 17 [E17]) from female pregnant mice (BALB/C, Charles River Laboratories International Inc., Wilmington, MA) and female pregnant rats (E17, Sprague Dawley [SD], bred-in-house) were used to isolate primary cortical neurons for in vitro primary culturing. Adult female SD rats (12–16 weeks old, bred-in-house) were used for in vivo studies to examine the BBB-permeability and neurotrophic effect of midi-GAGR. 12-month-old 3xTg-AD mice (female, B6; 129-Psen1tm1Mpm Tg [APPSwe, tauP301L] 1Lfa/Mmjax, Jackson Laboratory, Bar Harbor, ME) were used for the studies to examine the effects of midi-GAGR on neurotrophic and neurodegenerative markers in 3xTg-AD mouse brains. Animals were housed at room temperature under a 12-h light/dark cycle. Food and water were provided ad libitum. All experiments were performed during the light phase (7 am-7 pm). All the procedures of animal use described in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of University of Toledo College of Medicine and Life Science in accordance with National Institutes of Health guidelines.

Antibodies

Antibodies to FGFR1 (SAB4300488), neurofilament 200 (NF200, N4142), and α-tubulin (T9026), synaptophysin (S5768), and βIII-tubulin (T2200) were purchased from Sigma (St. Louis, MO). Antibodies to PSD95 (sc-32290), pCREB (P-Ser133, sc-7978), CREB (sc-377154), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32233), and growth associated protein 43 (GAP-43, sc-17790) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to PHF-tau (P-Ser202, AT8, MN1020B) was purchased from Thermo Scientific (Rockford, IL). Antibodies to Iba1 (ab5076) and pCREB (P-Ser133, ab30651) were purchased from Abcam Inc. (Cambridge, MA). Antibody to tau (610672) was purchased from BD Transduction Laboratories (Lexington, KY).

Hydrolysis of low acyl gellan gum

1 mL of 1% salicin solution (1 g salicin [Sigma] in 100 mL of 0.1 M acetate buffer [pH 5]) was pre-warmed at 37°C for 6–8 minutes and mixed with 2 mg of α(1→3)-glycosidase (Sigma) to make the enzyme solution for the hydrolysis of low acyl gellan gum (LA-GAGR, CPKelco Co. [Atlanta, GA]). The enzyme solution was diluted to 0.1 mg/mL before use. 0.48 g of LA-GAGR was dissolved in 80 mL of the acetate/salicin solution. 8 mL of LA-GAGR solution was mixed with 2 mL of the enzyme solution in 15 mL polypropylene cornical tubes and incubated at 37°C (80 rpm) for 24, 48, or 72 h for the enzymatic digestion of LA-GAGR. Enzyme reaction was stopped by incubation in a hot water bath for 5 min and dried in a vacuum dryer (-60 cm Hg gauge) at 70°C. Dried gel pellet was then washed extensively in de-ionized water by stirring for 48 h (fresh water replaced every 12 h) to wash off salts and enzyme from the pellet and processed for viscosity measurement using a parallel plate rheometer (PPR, Rheometrics, Inc., Piscataway, NJ) equipped with rheometer software, TA Orchestrator (TA Instrument, Inc., New Castle, DE). From the viscosity-storage modulus profiles, the MW distributions of the cleavage products were determined using the RheoAnalyzer program (TomCoat Oy, Inc., Finland). The pellet was dissolved in 100 mL de-ionized water, autoclaved at 120°C for 45 min, aliquoted, and kept at -80°C until use.

Drug treatment of Neuro2A (N2A) cells

N2A cells (passage 7–10, a gift from Dr. Marthe Howard at University of Toledo [ATCC^® CCL-131^™]) were sparsely seeded on coverslips in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Grand Island, NY) containing 10% heat-inactivated fetal bovine serum (FBS), 5 g/L D-glucose, 110 mg/L sodium pyruvate, and 1 x Pen Strep (Life Technologies) and incubated at 37°C in a humidified 5% CO₂ incubator for 48 h. Then, serum-containing medium was replaced with serum-free medium containing vehicle (H₂O) or different concentrations of midi-GAGR (0.001, 0.01, 0.1, 1, and 10 μM) and incubated for 3 days prior to immunocytochemistry using anti-α-tubulin antibody followed by secondary Alexa_488nm antibody (Life Technologies) [86]. Coverslips were then mounted on glass slides using Fluoromount G (Fisher Scientific, Pittsburgh, PA). The images of cells were taken using a TCS SP5 multi-photon laser scanning confocal microscope (Leica Microsystems, Bannockburn, IL). The confocal microscope is equipped with conventional solid state, a ti-sapphire tunable multi-photon laser (Coherent, Santa Clara, CA), and acousto optical beam splitter AOBS. Images were taken with either 40 x or 20 x Zeiss alpha plan fluor oil objective (1.4 NA). Cells having the neurite length longer than 2 times the diameter of cell body were chosen for image analysis. To examine the protective effect of midi-GAGR against oxidative stress-induced neurite atrophy, differentiated N2A cells were pre-treated with midi-GAGR (0, 0.001, 0.01, 0.1, 1, and 10 μM) and then with either 4-hydroxynonenal (4HNE) (Cayman Chemical, Ann Arbor, MI) or H₂O₂ (Sigma). First, we determined the toxic dose ranges of 4HNE and H₂O₂ that cause neurite atrophy by treating differentiated N2A cells with 0, 1, 5, 10 or 25 μM of 4HNE for 48 h or 0, 1, 10, 50, 100, or 200 μM of H₂O₂ for 24 h. Then, the doses that showed maximum inhibitory effects were used to treat differentiated N2A cells along with 0, 0.001, 0.01, 0.1, 1, and 10 μM of midi-GAGR. The total neurite lengths of N2A cells in different conditions were measured using ‘Metamorph’ software (Molecular Devices, Sunnyvale, CA) and used to calculate average total neurite lengths.

Drug treatment of primary rodent cortical neurons

We examined the protective effect of polysaccharides on primary cortical neurons from 4HNE, H₂O₂, and Aβ₄₂ peptide (Sigma) using LIVE/DEAD^® Viability/Cytotoxicity Assay Kit (Life Technologies). Female pregnant mice (BALB/C, E17, Charles River Laboratories International, Inc.) or female pregnant SD rats (E17, bred in house) were anesthetized and dissected to obtain 8–9 embryos per animal. Cortical neurons were isolated from embryonic brains and differentiated on poly-L-lysine-coated coverslips in B27/neurobasal medium. For drug treatment before free radical treatment, mouse cortical neurons (5 days in vitro [DIV5]) were treated with vehicle (H₂O), 1 μM of 5kD dextran (Sigma), alginate (Sigma), midi-GAGR, 0.01 μM of high acyl gellan gum (HA-GAGR, CPKelco Co.), or 0.1 μM of LA-GAGR for 24 h and then treated with 10 μM 4HNE or 50 μM H₂O₂ for 24 h, or 2 μM Aβ₄₂ peptide for 48 h prior to viability/cytotoxicity assay. For drug co-treatment with free radicals, rat cortical neurons (DIV5) were treated with 1 μM of dextran, alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or 0.1 μM of LA-GAGR along with either 10 μM 4HNE (24 h) or 2 μM Aβ₄₂ peptide (48 h). To examine the extent to which midi-GAGR-mediated neuroprotection depends on FGFR1, rat cortical neurons (E17, DIV5) were pre-treated with 4 μM SU5402 (Sigma) for 6 h and treated with 10 μM 4HNE and either vehicle or 1 μM midi-GAGR for 24 h prior to cell viability/cytotoxicity assay. As controls, neurons were treated with SU5402, midi-GAGR, 4HNE, 4HNE plus midi-GAGR, or SU5402 plus midi-GAGR.

For cell viability/cytotoxicity measurement, neurons on glass coverslips were incubated in 1 x PBS containing 2 μM calcein AM (live cells: green) and 4 μM ethidium homodimer-1 (dead cells: red) for 10 min at 37°C. Immediately thereafter, neurons were imaged by 10 x objective using a fluorescence Olympus IX71 microscope (Olympus America Inc., Center Valley, PA) and, for the acquisition of high-quality images, using TCS SP5 multi-photon laser scanning confocal microscope.

Co-culturing of microglial cells and primary rat cortical neurons

Microglial cell culture was prepared from the whole brain tissues (except of the cerebellum) of rat pups at postnatal day 3 (P3). Briefly, whole brain tissues except of the cerebellum were dissected and re-suspended in L-15 media on ice. Brain tissues were centrifuged at 1,000 x g for 3 min at 4°C. After the supernatant over brain tissue pellet was removed, the pellet was re-suspended in fresh L-15 media, followed by mechanical digestion using pasteur pipette. After the digestion, the resuspension was filtered through cell strainer (pore diameter = 70 μm). The flow-through was centrifuged at 1,000 x g for 3 min at 4°C. Then, cell pellet was re-suspended in DMEM media containing 10% FBS and 1X penicillin/streptomycin and plated at a high density in T75 culture flasks. The medium was exchanged with fresh medium every four days. After 8–10 days, culture flask caps were covered with parafilm to prevent gas exchange with environmental air. Flasks were then shaken in an orbital shaker at 220 rpm for 4 h at 37°C. Media was then collected into a conical tube and centrifuged at 800 x g for 10 min. The resulting cell pellet (mostly microglial cells) was then re-suspended in a neurobasal media containing B27 supplement and plated in filter insert (0.4-μm pore diameter) at a density of 2 x 10⁵ cells. The filter inserts containing microglial cells were transferred to a 24 well plate containing rat cortical neuron cultures (DIV6) at the bottom of each well. Microglial cells were treated with either 2 μM Aβ₄₂ peptide or vehicle (H₂O) and neurons with either 1 μM midi-GAGR or vehicle (H₂O) for 48 h prior to live-dead assay. In addition, we examined whether microglial cells penetrated the filter and fell down to neurons at the bottom of well or not by staining the cells on coverslips at the bottom of well by staining the coverslips with anti-Iba1 antibody for immunocytochemistry and confocal microscopy.

Analysis of neurite outgrowth and pCREB in primary mouse cortical neurons

To analyze the effect of polysaccharides on neurite outgrowth, primary mouse cortical neurons were treated with vehicle (H₂O) or 1 μM of dextran, alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or 0.1 μM of LA-GAGR for 2 days prior to immunocytochemistry using anti-α-tubulin antibody and secondary Alexa_488nm antibody. An etched grid coverslip containing 200 numbered boxes was used to select neurons objectively for image acquisition and analysis. A total of 24 boxes were randomly selected per treatment group. All the neurons having total neurite length longer than 4 times of the diameter of neuron cell body were chosen for image analysis. The total length of the neurites of each neuron was measured using Metamorph and used to calculate average total neurite length. To examine the effect of polysaccharides on the phosphorylation (activation) of nuclear CREB, neurons were stained with anti-pCREB antibody (Abcam & Santa Cruz biotechnologies, 2nd antibody with Alexa_568nm), anti-α-tubulin antibody (Sigma, 2nd antibody with Alexa_488nm), and DAPI (Sigma) after two-day incubation with vehicle (H₂O) or 1 μM of dextran, alginate, or midi-GAGR, 0.01 μM of HA-GAGR, or 0.1 μM of LA-GAGR. Then, to identify midi-GAGR-induced signaling pathway that induces CREB phosphorylation, primary mouse cortical neurons were pre-treated with the inhibitors of FGFR1 (SU5402 [Santa Cruz Biotechnologies], 4 μM), PKC (staurosporine [Sigma], 3 nM), MEK (U0126 [Sigma], 10 μM), PI3K (LY294002 [Sigma], 20 μM), CaMKII (KN-62 [Calbiochem, Billerica, MA], 10 μM), or FAK (PF-573228 [Sigma], 1 μM) for 6 h and then with 1 μM midi-GAGR for 48 h prior to the staining of pCREB and α-tubulin. The images of neurons on coverslips were taken by confocal microscopy at the same gain (850), offset (-0.01) and exposure time (2 sec). The intensity of the staining of nuclear pCREB was measured using Metamorph and used to calculate average intensity. In addition, we detected the phosphorylation of CREB in the cytosols of mouse cortical neurons treated with polysaccharides by immunoblotting. Primary mouse neurons were dissected from 16 mouse embryos (E17) and plated in the wells of 6-well plates (1 x 10⁶ cells/well), differentiated for 6 days, and treated with polysaccharides for 48 h. Then, neurons were harvested and lysed in 1% Igepal CA-630 (Sigma)-containing PMEE buffer plus protease and phosphatase inhibitor cocktails (Sigma) for protein extraction. Extracted proteins were separated in 4–12% NuPAGE Bis-Tris protein gels (Life Technologies) and transferred to nitrocellulose membrane (GE Healthcare Life Science, Pittsburg, PA) using a semidry blotter (Hoefer, Inc. San Francisco, CA). The protein bands on blots recognized by anti-pCREB (Santa Cruz Biotechnologies) and anti-CREB antibodies were detected on Amersham Hyperfilm^™ ECL films (GE Healthcare Life Science) using SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Scientific).

Examination of the in vivo neurotrophic effect of midi-GAGR

40 μL of 1 mM midi-GAGR or sterile H₂O (vehicle) was administered intranasally into the nostrils (20 μL/nostril) of SD rats (4 rats per each) using a pipette. Animals were kept in anesthetized condition using 4% isoflurane and at supine position during administration to prevent the squirting-out of drug. Animals were sacrificed by decapitation at 6 h, 24 h, or 48 h after the administration. Whole brain was micro-dissected into the frontal cortex, hippocampus, and the rest of brain. Then, tissues were homogenized in the 2-fold volume of 1 x PMEE buffer containing 1% Igepal CA-630 and protease inhibitor cocktail using a 2-mL Teflon homogenizer. The homogenization was then incubated on ice for 30 min at 4°C, followed by centrifugation at 14,500 x g for 30 min. The supernatant was collected and its protein concentration was measured by Bradford assay. 20 μg of proteins was loaded onto each well of NuPage 4–11% Bis-Tris protein gels. Immunoblotting was performed using the antibodies to NF200, GAP-43, and GAPDH. The densities of protein bands were measured using Image J and normalized to that of the loading control, GAPDH. Normalized values were used to calculate average normalized band densities.

Examination of the interaction of midi-GAGR with FGFR1 in brain synaptosomal plasma membrane

To examine whether midi-GAGR interacts with synaptosomal FGFR1 or not, we performed affinity chromatography using midi-GAGR-conjugated sepharose beads. We conjugated either midi-GAGR or dextran to epoxy-activated sepharose 6B that is a pre-activated medium that can be conjugated to the hydroxyl groups of carbohydrates. Briefly, 200 μL of 7.4 mM midi-GAGR or 5kD dextran in H₂O was mixed with 200 μL of epoxy-activated sepharose beads in a microtube and incubated on a rotator (16 h, 37°C). The mixture was spun down at 1,000 x g (10 min) to separate bead-bound polysaccharides from unbound. Unoccupied active sites on beads were blocked by incubation (4 h, 45°C) in 1 M ethanolamine (pH 8). Then, beads were washed with three cycles of alternating pH solutions– 0.1 M acetate buffer (pH 4) and 0.1 M Tris-HCl buffer (pH 8), both containing 0.5 M NaCl. The conjugation of polysaccharides to beads was confirmed by phenol—sulfuric acid colorimetry [87]. Cerebral cortices were dissected from four female mice (BALB/C, 8 wks old) and homogenized with a hand grinder in 1 mL of PMEE homogenization buffer plus 1% Igepal CA-630 and protease inhibitors (PIs). The homogenate was centrifuged at 1,000 x g (10 min) to remove nuclei and undisrupted cells. The supernatant was subjected to 5–6 strokes through 27G needle. Post-nuclear supernatant was centrifuged at 1,000 x g (15 min). The supernatant was diluted to 1:2 with Igepal CA-630-free PMEE buffer (to make 0.5% Igepal CA-630) and stored at -80°C until use. Then, the supernatant was incubated with 100 μL of either midi-GAGR-conjugated or dextran-conjugated beads. After 24-h incubation on a rotator at 4°C, the beads were washed three times with 0.5 mL of PMEE buffer to remove proteins that nonspecifically bind to beads. Then, the beads were boiled for protein elution in a SDS loading buffer. Eluted proteins were separated in a SDS-NuPAGE gel and processed for immunoblotting using FGFR1 antibody.

Examination of the effects of midi-GAGR on neuronal activity and neurodegenerative markers in 3xTg AD mice

We purchased 12-week-old 3xTg-AD mice (female, ~20 g, B6.Cg-Psen1tm1Mpm Tg [APPSwe, tauP301L]1Lfa/J) to examine the efficacy of midi-GAGR in restoring neuronal activity and reducing neurodegeneration in AD mouse brains. Three 3xTg AD mice were intranasally administered with either sterile H₂O (vehicle) or 1 mM midi-GAGR (40 μL total, 20 μL/nostril) every day for 14 days after 4% isoflurane anesthetization and then sacrificed by decapitation. Whole brain was micro-dissected to obtain the cortices and hippocampi. The tissues were homogenized in the 2-fold volume of 1 x PMEE buffer containing 1% Igepal CA-630 plus phosphatase and protease inhibitor cocktail using a miniature cell grinder for 1.5 mL microtube. The homogenization was then incubated on a rotator for 30 min at 4°C, followed by centrifugation at 14,500 x g for 30 min. The supernatant was collected and its protein concentration was measured by Bradford assay. 30 μg of proteins was loaded onto NuPage 4–11% Bis-Tris protein gels. Immunoblotting was performed using the antibodies to NF200, GAP-43, PSD95, synaptophysin, pCREB, CREB, p-tau (AT8), tau, and GAPDH. The densities of protein bands were measured using Image J and normalized to that of the loading control, GAPDH. Normalized values were used to calculate average normalized band densities.

Statistical Analysis

All cell culture experiments were replicated multiple times with different batches of cell cultures. Microscopic analysis was performed blindly by students. Statistical significance between two groups was calculated using unpaired student’s t-test with a value of p<0.05 that was considered statistically significant. Multiple comparisons were performed using one-way ANOVA followed by Dunnett’s or Bonferroni’s multiple comparisons tests (GraphPad Prism software, La Jolla, CA).

Generation of small-size polysaccharides from low acyl gellan gum

We had searched for a neuroprotective polysaccharide among those that are currently used in human as food additive. Then, we found the strong neuroprotective polysaccharide, low acyl gellan gum (LA-GAGR). 1 μM LA-GAGR protected differentiated N2A cells and their neurites from high doses of 4HNE (data not shown), a reactive lipid radical that causes neurodegeneration [88, 89]. In order to increase water solubility and diffuse-ability, we cleaved LA-GAGR into smaller sizes by enzymatic digestion (α(1→3)-glycosidase) for 24, 48, and 72 h. The MWs of its cleavage products were determined using Parallel Plate Rheometer that measures shear storage modulus and loss modulus and yields the viscosity profile of polysaccharides. From the viscosity-storage modulus profiles, the MW distributions of the cleavage products were determined using RheoAnalyzer program. The validity of the RheoAnalyzer program was verified by running polystyrene standard (NBS 706) on the Parallel Plate Rheometer and determining MW from the viscosity profile. The MW distributions of LA-GAGR and its 24-h, 48-h, and 72-h digestions are shown in ‘S1 and S2 Appendices’. The average MW of LA-GAGR was ~99,639 g/mole that is close to the value reported by CPKelco Co. The MWs of 24-h, 48-h, and 72-h cleavage products were ~30,245 g/mole, ~4,775 g/mole, and ~718 g/mole, respectively. The average MW of ~4,775 g/mole (named ‘midi-GAGR’) is equivalent to six repeating units and that of ~718 g/mole (named ‘mini-GAGR’) is to one repeating unit. Two small-size LA-GAGR products, midi-GAGR and mini-GAGR, were chosen for further examination regarding their neuroprotective effect.

Midi-GAGR rescues neurites from the atrophy caused by 4HNE and H₂O₂

We used differentiated N2A cells to examine if midi-GAGR and mini-GAGR protect neurites from the oxidative insults of 4HNE and H₂O₂ as LA-GAGR does. We, first, determined the atrophic dose ranges of the radicals by treating differentiated N2A cells with increasing concentrations of 4HNE and H₂O₂ for 48 h and 24 h, respectively. Treated cells were fixed, immunostained by anti-α-tubulin antibody, imaged by confocal microscopy, and examined regarding total neurite length. The average total neurite length of N2A cells was decreased in a dose-dependent manner in response to 4HNE and H₂O₂ to maximum extents at 25 μM 4HNE (Fig 2B and 2D) and 200 μM H₂O₂ (Fig 2C and 2E) compared to vehicle (Fig 2A). It suggests that 25 μM and 200 μM are the maximum doses of 4HNE and H₂O₂, respectively, that causes almost complete neurite atrophy in differentiated N2A cells. Then, we treated differentiated N2A cells with increasing concentrations of midi-GAGR prior to the treatment with either 25 μM 4HNE or 200 μM H₂O₂. Treatment with 0.1 and 1 μM midi-GAGR prior to 4HNE treatment rescued neurites up to ~70% of the control level (vehicle) (Fig 2F and 2H). Similarly, treatment with 0.1 and 1 μM midi-GAGR prior to H₂O₂ treatment rescued neurites up to ~100% of the control level (Fig 2G and 2I). We also examined the protective effect of mini-GAGR but found that mini-GAGR was not as potent as midi-GAGR in protecting neurites from the free radicals (data not shown). Therefore, we chose midi-GAGR that showed stronger neuroprotective effect against 4HNE and H₂O₂ for further study.

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Fig 2. Midi-GAGR reverses neurite atrophy caused by 4HNE and H₂O₂.

(A-E) Differentiated N2A cells were treated with different concentrations of 4HNE (0, 1, 5, 10 and 25 μM) for 48 h or H₂O₂ (0, 1, 10, 50, 100 and 200 μM) for 24 h and immunostained with α-tubulin antibody. The representative images of N2A cells treated with H₂O (vehicle) (A), 25 μM 4HNE (B), or 200 μM H₂O₂ (C). Scale bar = 75 μm. Bar graphs represent the average total neurite lengths of N2A cells in response to different concentrations of either 4HNE (D) or H₂O₂ (E). *, p < 0.05 and **, p < 0.001 compared to control. (F-I) Differentiated N2A cells were pre-treated with different concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of midi-GAGR for 24 h, followed by incubation with either 25 μM 4HNE for 48 h or 200 μM H₂O₂ for 24 h and then immunostained with anti-α-tubulin antibody. The representative images of N2A cells pretreated with 1 μM midi-GAGR and then incubated with either 25 μM 4HNE (F) or 200 μM H₂O₂ (G). Scale bar = 75 μm. Bar graphs show the average total neurite lengths of N2A cells pre-treated with different concentrations of midi-GAGR followed by treatment with 25 μM 4HNE (H) or 200 μM H₂O₂ (I). Dotted lines correspond to the average total neurite lengths of N2A cells without any treatment. Data represent mean ± SEM of, at least, 40 cells/group from two independent experiments. *, p<0.05 and **, p<0.001 compared to 4HNE alone.

https://doi.org/10.1371/journal.pone.0149715.g002

Midi-GAGR reduces the apoptosis of rodent cortical neurons caused by 4HNE, H₂O₂, and amyloid β peptide

Based on our observation that midi-GAGR attenuated neurite atrophy caused by free reactive radicals, we speculated that midi-GAGR might protect neurons from the death caused by free radical insults. Thus, we examined the extent to which midi-GAGR protects the primary culture of rodent cortical neurons from 4HNE and H₂O₂. In addition to the radicals, we tested amyloid β peptide (Aβ₄₂) because Aβ₄₂ peptide is a major causative factor that causes oxidative stress and neuron death [90, 91]. We treated mouse cortical neurons (E17, DIV5) with 10 μM 4HNE (24 h), 50 μM H₂O₂ (24 h), or 2 μM Aβ₄₂ peptide (48 h) after the treatment of the neurons with vehicle (H₂O), midi-GAGR (1 μM), dextran (1 μM), alginate (1 μM), LA-GAGR (0.1 μM), or HA-GAGR (0.01 μM) for 24 h. The concentrations of 4HNE, H₂O₂, and Aβ₄₂ peptide were chosen according to their patho-physiological concentrations [89, 92–95]. The concentrations of LA-GAGR and HA-GAGR were chosen because, within the same volume, the total numbers of sugar units in the polysaccharides at the concentrations are close to that of 1 μM midi-GAGR. The viability of neurons was assessed using LIVE/DEAD^® Viability/Cytotoxicity Assay Kit in which membrane-permeant calcein AM is cleaved by esterase in live cells, thus yielding green fluorescence, and membrane-impermeant ethidium homodimer-1 stains the nucleic acids of plasma membrane-compromised cells with red fluorescence. We counted the numbers of green (live) and red (dead) neurons in each condition using Metamorph. Although the intensities of green fluorescent signals in neurons treated with either free radicals or Aβ₄₂ peptide were weak, we included the neurons in our counting. About 8–9% of vehicle-treated neurons died during the process of live/dead cell assay (Figs 3A and 4). Upon exposure to 10 μM 4HNE, ~26% of mouse cortical neurons died (Figs 3B and 4A) while pre-treatment with 1 μM midi-GAGR (Fig 3C) and 0.1 μM LA-GAGR reduced neuron death to 14% and 10%, respectively (Fig 4A). HA-GAGR and alginate also reduced the percent of neuron death caused by 4HNE while dextran did not (Fig 4A). Exposure to 50 μM H₂O₂ caused neuron death in ~25% of cortical neurons that were pre-treated with vehicle (Figs 3D and 4B). Pre-treatment with either alginate or dextran did not reduce H₂O₂-caused neuron death (Fig 4B). Conversely, pre-treatment with midi-GAGR (Fig 3E), LA-GAGR, or HA-GAGR reduced neuronal death to ~13% (Fig 4B). These results suggest that midi-GAGR, LA-GAGR, and HA-GAGR can protect rodent cortical neurons from both H₂O₂ and 4HNE while dextran and alginate cannot.

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Fig 3. Pre-treatment with midi-GAGR significantly reduces the apoptosis of rodent cortical neurons caused by 4HNE, H₂O₂, and amyloid β peptide.

(A-G) Mouse cortical neurons at DIV5 were pre-treated with H₂O (vehicle), 1 μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA-GAGR, 1 μM alginate or 1 μM dextran followed by incubation with vehicle, 10 μM 4HNE, or 50 μM H₂O₂ for 24 h or 2 μM Aβ₄₂ for 48 h. After the treatment, neurons were processed for live/dead assay using calcein AM and ethidium homodimer-I. The representative images of H₂O (A), 4HNE alone (B), 4HNE + midi-GAGR (C), H₂O₂ alone (D), H₂O₂ + midi-GAGR (E), Aβ₄₂ alone (F), and Aβ₄₂ + midi-GAGR (G). Live cells were labeled as green and dead cells as red. Scale bar = 100 μm. Insets show the magnified images of individual neurons. Inset scale bar = 50 μm.

https://doi.org/10.1371/journal.pone.0149715.g003

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Fig 4. Quantification of neuron death in response to oxidative insults in the presence of different polysaccharides.

The numbers of live and dead cells were counted using Metamorph software. Bar graphs represent the percents of dead neurons after the pre-treatments with different polysaccharides followed by treatment with 4HNE (A), H₂O₂ (B), or Aβ₄₂ (C). Data represent mean ± SEM of three independent experiments. For each experiment, at least, 1,000 cells per group were counted. *, p<0.05.

https://doi.org/10.1371/journal.pone.0149715.g004

We, then, examined the extent to which midi-GAGR protects cortical neurons from Aβ₄₂ peptide. Exposure to 2 μM Aβ₄₂ peptide caused the death of about 30% of neurons pre-treated with vehicle (Figs 3F and 4C). Pre-treatment with HA-GAGR, alginate, or dextran did not decrease the percent of neuron death compared to pre-treatment with vehicle (Fig 4C). Conversely, pre-treatment with either midi-GAGR or LA-GAGR reduced neuron death to ~10%, the control level (Figs 3G and 4C). Thus, only midi-GAGR and LA-GAGR can protect cortical neurons from Ab₄₂ peptide while other polysaccharides cannot.

Midi-GAGR reduces the apoptosis of rodent cortical neurons from co-treated 4HNE or Aβ₄₂ peptide

It is likely that free reactive radicals already exist at pathological concentrations inside the brain before some symptoms are noticeable for treatment. In other words, treatment is likely to start at the pre-existence of the pathological concentrations of the radicals. Thus, we examined if midi-GAGR can protect cortical neurons from co-treated 4HNE or Aβ₄₂ peptide. Rat cortical neurons (E17, DIV5) were treated with either 10 μM 4HNE (24 h) or 2 μM Aβ₄₂ peptide (48 h) and either vehicle or 1 μM midi-GAGR. Then, the viability of neurons was assessed using LIVE/DEAD^® Viability/Cytotoxicity Assay Kit. Exposure to 10 μM 4HNE caused apoptosis in ~27% of vehicle-treated neurons while co-treatment with midi-GAGR reduced the percent of neuron death to ~9% (Fig 5A). Treatment with 2 μM Aβ₄₂ peptide caused death in ~29% of vehicle-treated neurons while co-treatment with midi-GAGR reduced the percent of neuron death to ~ 15% (Fig 5A). These results suggest that midi-GAGR can also protect rodent cortical neurons from co-treated 4HNE or Aβ₄₂ peptide.

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Fig 5. Midi-GAGR protects rodent cortical neurons from co-treated 4HNE, Aβ₄₂ peptide and activated microglial cells.

(A) Rat cortical neurons (E17) at DIV5 were co-treated with either 10 μM 4HNE (for 24 h) or 2 μM Aβ₄₂ (for 48 h) and either water or 1 μM midi-GAGR. After the treatment, neurons were processed for live/dead assay using calcein AM and ethidium homodimer-I. Live and dead cells were imaged using a fluorescence microscope. The numbers of live and dead cells were counted using Metamorph software. Bar graphs show the percents of dead neurons after co-treatment with either 4HNE or Aβ₄₂ plus/minus midi-GAGR. Data represent mean ± SEM of three independent experiments. *, p<0.05. (B) The co-cultures of rat cortical neurons and microglia cells were treated with 2 μM Aβ₄₂ plus/minus 1 μM midi-GAGR. After 48 h, transwell filters containing microglial cells were removed and neurons in bottom wells were processed for live/dead assay. Live and dead cells were imaged using a fluorescence microscope. The numbers of live and dead neurons were counted using Metamorph software. Bar graphs show the percent of dead neurons. Data represent mean ± SEM of three independent experiments. *, p<0.05.

https://doi.org/10.1371/journal.pone.0149715.g005

Midi-GAGR protects rodent cortical neurons from microglial cells activated by Aβ₄₂ peptide

Microglial cells activated by Aβ₄₂ peptide are a major causative factor for neurodegeneration, especially AD [96]. As such, activated microglial cells secrete free radicals and pro-inflammatory cytokines that facilitate neurodegeneration and neuron apoptosis. Thus, we examined if midi-GAGR protects primary rodent cortical neurons from microglia cells activated by Aβ₄₂ peptide. Microglial cells were isolated from rats on the postnatal day 1 (S3 Appendix) and seeded in 0.4μm-pore-size filter insert that fits into the well of 24-well plate. The filter inserts were transferred to a 24-well plate in which primary rat cortical neurons (E17) were cultured at the bottoms of wells for 6 days (DIV6). Then, microglia cells were treated with 2 μM Aβ₄₂ and neurons with either vehicle or 1 μM midi-GAGR. After 48 h, the viability of neurons was assessed using LIVE/DEAD Viability/Cytotoxicity Assay Kit. Around 25% of the neurons treated with vehicle died under untreated microglial cells (Fig 5B). Upon treatment of microglial cells with Aβ₄₂ peptide, the percent of death in neurons treated with vehicle was increased to ~40%. Conversely, treatment of neurons with 1 μM midi-GAGR reduced death to ~27% that was close to the percent of dead neurons under untreated microglial cells (Fig 5B). Thus, it is clear that midi-GAGR can protect rodent cortical neurons from activated microglial cells.

Midi-GAGR enhances neurite outgrowth in N2A cells and rodent cortical neurons

In addition to the neuroprotective effect, we found that LA-GAGR enhanced neurite outgrowth in N2A cells. Thus, we expected that midi-GAGR would have a similar enhancing effect on neuritogenesis. First, we examined the neuritogenic effect of midi-GAGR on N2A cells. N2A cells were differentiated with increasing concentrations (0, 0.001, 0.01, 0.1, 1, and 10 μM) of midi-GAGR for 48 h. Then, cells were fixed and immunostained with anti-α-tubulin antibody. The total length of neurites per cell was measured using Metamorph and used to calculate average total neurite length per treatment group. At 0.1 and 1 μM, the average total neurite length of midi-GAGR-treated N2A cells reached ~1.7 fold higher than that of vehicle-treated cells (Fig 6; 438.21±20.55 μm [0.1 μM] and 457.76±41.66 μm [1 μM] vs. 257.51±45.16 μm [vehicle], p<0.05). At 10 μM, average total neurite length was decreased, which is similar to the pattern of neuritogenesis in cells treated with FGL [79]. We also examined the neuritogenic effect of midi-GAGR on primary mouse cortical neurons (E17, DIV4). Neurons were incubated with vehicle, midi-GAGR (1 μM), dextran (1 μM), alginate (1 μM), LA-GAGR (0.1 μM), or HA-GAGR (0.01 μM) for 2 days and immunostained with the antibody to βIII tubulin (Fig 7A–7F). The total neurite length of each neuron was measured to calculate average total neurite length per condition. Then, average total neurite length per condition was divided by that of vehicle treatment to obtain fold change in average total neurite length per condition. Compared to vehicle treatment (Fig 7A), midi-GAGR (Fig 7B) and LA-GAGR (Fig 7C) increased average total neurite length by ~1.6 fold (Fig 7G). Conversely, HA-GAGR (Fig 7D), alginate (Fig 7E), and dextran (Fig 7F) did not enhance neuritogenesis (Fig 7G). Thus, only midi-GAGR and LA-GAGR have strong neuritogenic effect on rodent cortical neurons.

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Fig 6. Midi-GAGR enhances neurite outgrowth in N2A cells.

Differentiated N2A cells were treated with different concentrations of midi-GAGR for 48 h and immunostained using anti-α-tubulin antibody. Representative confocal images of N2A cells treated with either H₂O (vehicle) (A) or 1 μM midi-GAGR (B). Scale bar = 30 μm. (C) Bar graphs show the average total neurite lengths of N2A cells treated with different concentrations of midi-GAGR (mean ± SEM of, at least, three independent experiments). *, p<0.05 compared to control.

https://doi.org/10.1371/journal.pone.0149715.g006

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Fig 7. Midi-GAGR enhances neuritogenesis in mouse cortical neurons.

(A-F) Mouse cortical neurons (E17, DIV4) were treated with H₂O (vehicle) (A), 1 μM midi-GAGR (B), 0.1 μM LA-GAGR (C), 0.01 μM HA-GAGR (D), 1 μM alginate (C), or 1 μM dextran (F) for 48 h and immunostained with anti-α-tubulin antibody. Scale bar = 100 μm. (G) Bar graphs show average fold changes in the total neurite length of mouse cortical neurons in response to different polysaccharides (mean ± SEM of, at least, three independent experiments). **, p<0.01 compared to control.

https://doi.org/10.1371/journal.pone.0149715.g007

Midi-GAGR activates CREB, a neurotrophic transcriptional factor

Since both survival-enhancement and neuritogenesis converge on neurotrophic effect [33, 97], it is possible that midi-GAGR activates a neurotrophic signaling pathway that increases neuron survival in neurodegenerative condition and enhances neuritogenesis. To examine the possibility, we stained midi-GAGR-treated mouse cortical neurons with the antibody to pCREB, a marker for activated neurotrophic signaling pathways [98–100]. Mouse cortical neurons (E17, DIV4) were treated with vehicle (Fig 8A), midi-GAGR (1 μM, Fig 8B), alginate (1 μM, Fig 8C), dextran (1 μM, Fig 8D), LA-GAGR (0.1 μM), or HA-GAGR (0.01 μM) for 48 h, fixed, immunostained with the antibodies to α-tubulin and pCREB along with DAPI. The fluorescence intensity (arbitrary number) of stained pCREB in the nucleus was measured using Metamorph. Vehicle-treated neurons showed the basal levels of pCREB in the nuclei (Fig 8A). Conversely, treatment with either midi-GAGR (Fig 8B) or LA-GAGR significantly increased the level of nuclear pCREB while the other polysaccharides did not (Fig 8C and 8D). Treatment with HA-GAGR slightly increased the level of nuclear pCREB. We calculated the average intensity of stained pCREB in the nuclei of the neurons. Treatment with either midi-GAGR or LA-GAGR increased the average intensity of nuclear pCREB by ~2 fold compared to vehicle treatment (Fig 8E). To confirm the result of the optical measurement of the level of pCREB, we performed immunoblotting to detect the phosphorylation of CREB protein using the cytosols extracted from mouse cortical neurons treated with vehicle or polysaccharides. Total CREB was also detected by immunoblotting. Compared to control (vehicle treatment), the levels of pCREB were significantly increased in neurons treated with either midi-GAGR or LA-GAGR while those in neurons with either alginate or dextran were not (Fig 8F). The phosphorylation level of HA-GAGR was also increased. Thus, midi-GAGR and LA-GAGR increase the phosphorylation of CREB significantly and HA-GAGR does slightly.

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Fig 8. Midi-GAGR activates CREB, a neurotrophic transcriptional factor.

Mouse cortical neurons (DIV4) were treated with H₂O (vehicle), 1 μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA GAGR, 1 μM alginate, or 1 μM dextran for 48 h and immunostained with DAPI (not shown) and the antibodies to α-tubulin and pCREB. The representative images of neurons treated with H₂O (A), midi-GAGR (B), alginate (C) or dextran (D), followed by staining with α-tubulin (green) and pCREB (red). Scale bar = 30 μm. (C) Bar graphs show the average intensities of pCREB after different treatments (n = 90 neurons, mean ± SEM of, at least, three independent experiments). *, p<0.05 compared to control. (D) The cytosols extracted from neurons treated with H₂O (vehicle), 1 μM midi-GAGR, 0.1 μM LA-GAGR, 0.01 μM HA GAGR, 1 μM alginate, or 1 μM dextran for 48 h were used for immunoblotting using the antibodies to pCREB and CREB (neurons extracted from sixteen E17 mouse embryos, n = 2 experiments).

https://doi.org/10.1371/journal.pone.0149715.g008

Intranasally administered midi-GAGR penetrates the BBB and increases the expression of NF200 and GAP-43 in the frontal cortex and hippocampus

Our previous study demonstrated that midi-GAGR penetrated the BBB and remained at 0.1–0.2 μM minside the brain for ~24 h after one-time intranasal administration [101]. Therefore, intranasally administered midi-GAGR is expected to exert a neurotrophic effect inside the brain within 24 h post-administration. We examined the expression of two neurotrophic protein markers, NF200 and GAP-43, in the brains of rats administered intranasally with 40 μL of either H₂O or 1 mM midi-GAGR. At 6, 24, and 48 h after the intranasal administration, the frontal cortex, hippocampus, and the rest of the brain were dissected from the rats and processed for immunoblotting using the antibodies to NF200, GAP-43, and GAPDH (loading control). The protein band densities of NF200 and GAP-43 were measured by densitometry (Image J), normalized by those of GAPDH, and used to calculate average normalized values. In the frontal cortex, the expression level of NF200 was increased to the level significantly higher than control at 6 h and 24 h after the administration (Fig 9A and 9B). In the hippocampus, that of NF200 was increased to the level statistically higher than control after 24 h. The expression level of GAP-43 was also significantly increased in the frontal cortex at 6 h and 24 h while slightly increased in the hippocampus at 24 h (Fig 9A and 9C). The rest of the brain did not show an increase in NF200 and GAP-43 after one-time intranasal administration. These results indicate that intranasally administered midi-GAGR enters the brain and exerts its neurotrophic effect in the frontal cortex and hippocampus within 24 h post-administration.

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Fig 9. Intranasally administered midi-GAGR increases the expression of NF200 and GAP-43 in the brains of live rats.

SD rats were intranasally administered with either vehicle or midi-GAGR and processed to obtain brains at 6h, 24h or 48h after the administration. Brains were dissected to the frontal cortex, hippocampus, and rest of the brain. (A) Brain tissue lysates were processed for immunoblotting using the antibody to NF200 (upper panel), GAP-43 (middle panel), or GAPDH (lower panel). ‘C’ is control and ‘M’ is midi-GAGR. The band densities of NF200 and GAP-43 were measured using image J software and normalized to those of GAPDH. Bar graphs show fold changes in the level of NF200 (B) and GAP-43 (C) in the different parts of brains at given time points. Data represents mean ± SEM (n = 4 animals/group). *, p<0.05.

https://doi.org/10.1371/journal.pone.0149715.g009

Midi-GAGR binds to FGFR1 and activates FGFR1 signaling pathway

Our pilot proteomics study hinted that midi-GAGR might interact with FGFR1 (data not shown). Thus, we examined the possible interaction of midi-GAGR with FGFR1 by affinity chromatography using midi-GAGR-conjugated sepharose column. Either midi-GAGR or dextran was conjugated to epoxy sepharose beads according to manufacturer’s protocol. Whole mouse brains were homogenized for cytosol extraction in PMEE buffer containing 1% Igepal CA-630 and protease inhibitor cocktail. Brain cytosols were diluted to 1:2 to make 0.5% Igepal buffer prior to the incubation with either dextran- or midi-GAGR-conjugated sepharose beads on a rotating plate for 16 h at 4°C. Nonspecific bindings were removed by extensive washes in PMEE buffer. Beads were boiled for protein elution. FGFR1 was one of the proteins eluted from midi-GAGR beads but not from dextran beads (Fig 10A).

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Fig 10. Midi-GAGR binds to FGFR1 and uses FGFR1 signaling pathway to activate CREB and protect neurons from the death caused by oxidative insult.

(A) Midi-GAGR- or dextran-conjugated epoxy sepharose beads were mixed with synaptosomal plasma membrane proteins in 0.5% Igepal CA-630 PMEE buffer to pull down midi-GAGR-interacting FGFR1. Precipitated FGFR1 was detected by immunoblotting (n = 2, four rat brains). (B-J) Mouse cortical neurons (DIV4) were pre-treated with H₂O (vehicle, B,C) or the inhibitors of FGFR1 (SU5402 [SU], 4 μM, D), PKC (staurosporine [Stau], 3 nM, E), MEK (U0126 [U01], 10 μM, F), PI3K (LY294002 [LY], 20 μM, G), CaMKII (KN-62 [KN], 10 μM, H), or PF-573228 (PF, 1 μM, I) for 6 h and then with mock (B) or 1 μM midi-GAGR (+midi, C-I) for 48 h. Neurons were then immunostained with the antibodies to α-tubulin (red) and p-CREB (green). Scale bar = 100 μm. (J) Bar graphs show the average intensities of pCREB after different treatments (n = 60 neurons, mean ± SEM). *, p<0.01 and #, p<0.05 compared to control. (K) Rat cortical neurons (E17, DIV6) were treated for 6 h with FGFR1 inhibitor (SU5402, 4 μM) and treated with 10 μM 4HNE and either vehicle or 1 μM midi-GAGR for 24 h prior to cell viability/cytotoxicity assay. As controls, neurons were treated with 4HNE, 4-HNE plus midi-GAGR, SU5402, midi-GAGR, or SU5402 plus midi-GAGR. Live and dead cells were imaged using a fluorescence microscope. The numbers of live and dead neurons were counted using Metamorph software. Bar graphs show the percent of dead neurons. Data represent mean ± SEM of three independent experiments. For each experiment, at least, 200 cells per group were counted. *, p<0.05 (n.s.: not significant).

https://doi.org/10.1371/journal.pone.0149715.g010

As midi-GAGR binds to FGFR1, midi-GAGR is expected to activate FGFR1 signaling pathway, which results in CREB phosphorylation. We used pharmacological agents to inhibit signaling molecules downstream of FGFR1 that mediate CREB phosphorylation. We measured the fluorescence intensities of nuclear pCREB in mouse cortical neurons (E17, DIV6) pre-treated for 6 h with the inhibitors of FGFR1 (SU5402 [SU], 4 μM), PKC (staurosporine [Stau], 3 nM), MEK (U0126 [U01], 10 μM), PI3K (LY294002 [LY], 20 μM), or CaMKII (KN-62 [KN], 10 μM) and then with 1 μM midi-GAGR (midi) for 48 h. PF-573228 (PF, 1 μM) that inhibits FAK which sits at the bottleneck of the signaling pathway downstream of NCAM180 [73] was also included to examine whether NCAM180 is involved in midi-GAGR-mediated CREB phosphorylation or not. Treated neurons were stained with the antibodies to βIII tubulin (red) and pCREB (green) for the measurement of the average intensities of nuclear pCREB. Compared to neurons treated with vehicle (Fig 10B and 10J), those with midi-GAGR (midi) showed a significant increase (~2.6 fold) in the average intensity of pCREB (Fig 10C and 10J). Conversely, pre-treatment with the inhibitor of FGFR1 (Fig 10D and 10J), PKC (Fig 10E and 10J), or MEK (Fig 10F and 10J) significantly decreased the average intensity of pCREB (solid lines, *: p<0.01, n = 40–50 neurons). In neurons pre-treated with the inhibitor of either PI3K or CaMKII, the average intensity of pCREB was decreased slightly but still statistically significantly (dotted lines, #: p<0.05, n = 40–50 neurons). In contrast to the inhibitors, pre-treatment with FAK inhibitor did not decrease the average intensity of pCREB in midi-GAGR-treated neurons (Fig 10I and 10J). These results suggest that midi-GAGR activates FGFR1 and its downstream signaling pathways consisting of PKC, MEK, PI3K, and CaMKII, but not NCAM180-FAK signaling pathway for CREB phosphorylation.

Next, we examined how much FGFR1-mediated neurotrophic signaling pathway contributes to midi-GAGR-mediated neuroprotection against oxidative insult. We pre-treated rat cortical neurons (E17, DIV6) for 6 h with FGFR1 inhibitor (SU5402, 4 μM) and then with 10 μM 4HNE for 24 h prior to LIVE/DEAD Viability/Cytotoxicity Assay. Treatment with 4HNE increased the percent of dead cells from ~20% to ~60% while co-treatment with 1 μM midi-GAGR decreased that to ~30% (Fig 10K). Treatment with SU5402 alone or SU5402 plus midi-GAGR did not increase the percent of dead cells (Fig 10K). Interestingly, pre-treatment with SU5402 significantly increased the percent of dead cells up to ~80% upon the post-treatment with 4HNE (Fig 10K). In neurons pre-treated with SU5402 and then treated with 4HNE, midi-GAGR could not decrease the percent of dead cells. This result indicates that FGFR1-mediated signaling pathway plays a major role in midi-GAGR-mediated neuroprotection against oxidative insult.

Intranasally administered midi-GAGR increases neuronal activity markers and reduces hyperphosphorylated tau in 3xTg-AD mice

Given that the activation of FGFR1 enhances neuronal activity and, possibly, attenuates neurodegeneration [51–53], we speculate that midi-GAGR treatment does the similar. Thus, we examined if midi-GAGR treatment increases the protein markers of neuronal activity and reduces the neurodegeneration marker, hyperphosphorylated tau (P-Ser202), in AD brain [102]. We used 12-month-old 3xTg-AD mice that harbor two familial AD mutations, APP_swe and PS1_M146V, and the tau_P301L mutation found in frontotemporal dementia [103, 104]. Until 12 months of age, 3xTg-AD mice [103] develop synapse loss, Aβ peptide accumulation [105, 106], memory deficit [107], and tau hyperphosphorylation [103]. We used female 3xTg-AD mice since females show more obvious cognitive defects than males [108–110]. We administered 40 μL of vehicle (sterile H₂O) or 1 mM midi-GAGR intranasally into female 3xTg-AD mice once per day for 14 days. During midi-GAGR administration, 3xTg-AD mice did not show any abnormal behavior. Of note, 3xTg-AD mice administered with midi-GAGR made tight nest every day while those with vehicle made loose nest as previously reported [111]. After 14-day administration, mice were killed for brain extraction. Extracted brains were dissected to obtain the cortices and hippocampi. The brain tissues were lysed in PMEE buffer containing 1% Igepal CA-630 and phosphatase and protease inhibitor cocktails for immunoblotting using antibodies to NF200, GAP-43, PSD95, synaptophysin (SYN), pCREB, CREB, p-tau (AT8), tau, and GAPDH. The protein band densities of detected proteins were normalized by those of GAPDH and used to calculate average normalized band density for each protein. Compared to 3xTg-AD mice administered with vehicle (Veh.), NF200 was increased significantly in the hippocampus of those with midi-GAGR while not changed in the cortex (Fig 11A and 11B). GAP-43 and PSD95, the postsynaptic markers for increased synaptic activity, were increased significantly in both cortex and hippocampus of 3xTg-AD mice treated with midi-GAGR compared to control while synaptophysin (SYN) remained unchanged (Fig 11A, 11C, 11D and 11E). pCREB, another postsynaptic marker that shows increased neurotrophic signaling, was also significantly increased in both cortices and hippocampi of 3xTg-AD mice treated with midi-GAGR compared to control (Fig 11A and 11F). Total CREB was slightly increased in the hippocampus of midi-GAGR-treated 3xTg-AD mice while not changed in the cortex (Fig 11A and 11G). Surprisingly, hyperphosphorylated tau (P-Ser202) was drastically decreased in the hippocampus of 3xTg-AD mice and slightly in their cortices (Fig 11A and 11H). Total tau was not changed (Fig 11A and 11I). These results indicate that intranasally administered midi-GAGR not only enhances neuronal and synaptic activities in the cortex and hippocampus but also decreased hyperphosphorylated tau, a major AD facilitator, in the brains of 3xTg-AD mice.

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Fig 11. Intranasally administered midi-GAGR increased neuronal activity markers and decreased hyperphosphorylated tau in 3xTg-AD mice.

(A) 12-week-old 3xTg-AD mice were intranasally administered with 40 μL (20 μL/nostril) of either sterile H₂O (Veh.) or 1 mM midi-GAGR (midi) every day for 14 days and processed to obtain the cortex and hippocampus (Hippo). The tissues were homogenized to extract proteins for immunoblotting using the antibodies to NF200, GAP-43, PSD95, synaptophysin, pCREB, CREB, p-tau (AT8), tau, and GAPDH. (B-J) The densities of protein bands were measured using Image J and normalized to that of the loading control, GAPDH. Normalized values were used to calculate the average normalized band densities of NF200 (B), GAP-43 (C), PSD95 (D), synaptophysin (SYN, E), pCREB (F), total CREB (G), p-tau (H), and total tau (I). Data represent mean ± SEM of three independent animals. #, p<0.01, *, p<0.05.

https://doi.org/10.1371/journal.pone.0149715.g011