Herbs and Helpers
• 1Division of Neuromedical Science, Institute of Natural Medicine, University of Toyama, Toyama, Japan
• 2R&D Center, Kobayashi Pharmaceutical Co., Ltd., Ibaraki, Japan
Memory impairments in Alzheimer’s disease (AD) occur due to degenerated axons and disrupted neural networks. Since only limited recovery is possible after the destruction of neural networks, preventing axonal degeneration during the early stages of disease progression is necessary to prevent AD. Polygalae Radix (roots of Polygala tenuifolia; PR) is a traditional herbal medicine used for sedation and amnesia. In this study, we aimed to clarify and analyze the preventive effects of PR against Memory deficits in a transgenic AD mouse model, 5XFAD. 5XFAD mice demonstrated memory deficits at the age of 5 months. Thus, the water Extract of Polygalae Radix (PR extract) was orally administered to 4-month-old 5XFAD mice that did not show signs of memory impairment. After consecutive administrations for 56 days, the PR extract prevented cognitive deficit and axon degeneration associated with the accumulation of amyloid β (Aβ) plaques in the perirhinal cortex of the 5XFAD mice. PR extract did not influence the formation of Aβ plaques in the brain of the 5XFAD mice. In cultured neurons, the PR extract prevented axonal growth cone collapse and axonal atrophy induced by Aβ. Additionally, it prevented Aβ-induced endocytosis at the growth cone of cultured neurons. Our previous study reported that endocytosis inhibition was enough to prevent Aβ-induced growth cone collapse, axonal degeneration, and memory impairments. Therefore, the PR extract possibly prevented axonal degeneration and memory impairment by inhibiting endocytosis. PR is the first preventive drug candidate for AD that inhibits endocytosis in neurons.
Alzheimer’s disease is a progressive, degenerative, and irreversible neurological disorder. Although several clinical drugs are available for AD patients, these drugs only moderate the progression of AD. None of the AD treatments have succeeded in recovering cognitive function once the disease has progressed. Thus, since it is too late to treat AD with severe symptoms, a therapy or the prevention of AD at an early stage or before the onset of symptoms is required. Memory deficits are one of the most important core features of AD. Although AD can develop due to heterogeneous risk factors, such as genome, epigenome, and environments (Killin et al., 2016; Maloney and Lahiri, 2016; Pimenova et al., 2017), the common features of AD include amyloid β (Aβ)-induced degeneration of neurites, disruption of neural networks, and memory deficits (Dickson and Vickers, 2001; Hardy and Selkoe, 2002; Perl, 2010; Selkoe and Hardy, 2016). Preventing axonal degeneration could inhibit the progression of memory deficits in AD (Dickson and Vickers, 2001; Hardy and Selkoe, 2002; Simmons et al., 2014). Additionally, axonal regeneration is related to recovery from cognitive dysfunction in murine models of AD (Tohda, 2016).
Polygalae Radix (roots of Polygala tenuifolia; PR) is a traditional herbal medicine, which has been clinically used for memory loss in East Asia (Wu et al., 2011; May et al., 2013). There are many reports about the anti-AD properties of PR. For example, PR extracts and their constituents were reported to have a protective effect in cultured neurons from Aβ-induced neurotoxicity (Xu et al., 2011); they enhanced axon elongation when cultured neurons were already undergoing Aβ-induced axon atrophy (Naito and Tohda, 2006), inhibited Aβ secretion in cultured cells (Jia et al., 2004; Lv et al., 2009), and recovered memory in intrahippocampally Aβ-injected mice (Xu et al., 2011; Liu et al., 2015). Additionally, BT-11, an extract of PR, was reported to recover memory in stress- or scopolamine-induced amnestic rats (Park et al., 2002; Shin et al., 2009), and enhance memory in healthy humans (Lee et al., 2009). Taking these reports into consideration, PR may also have preventive effects against AD. At present, however, there are no studies that have demonstrated that PR prevents the development of AD in humans or animal models.
In AD, the increases in the level of Aβ in the brain precede memory deficits (Jack et al., 2010). A similar phenomenon is also observed in a transgenic mouse model of AD, 5XFAD mice, which expresses mutant human amyloid precursor protein (the Swedish mutations: K670N and M671L; the Florida mutation: I716V; and the London mutation: V717I) and PS1 (M146L; L286V) transgenes, under the neuron-specific mouse Thy-1 promoter (Oakley et al., 2006). Aβ plaques accumulate in the brains of 5XFAD transgenic mice by the age of 2 months, and memory impairments by 4-5 months (Oakley et al., 2006) and axon degeneration by 4-7 months (Tohda et al., 2011, 2012; Yang et al., 2017). This corroborated with the trend seen in patients with AD (Benes et al., 1991; Masliah et al., 1993). In the current study, PR water extract was consecutively administered before the onset of memory impairments in the 5XFAD mice, and its preventive effects on axonal degeneration and memory impairment were investigated. We focused on endocytosis as its inhibition is sufficient to prevent Aβ-induced axonal degeneration and memory deficits, and cause these preventive effects (Kuboyama et al., 2015).
Materials and Methods
Polygalae Radix Extract
The dried powder of the water extract of Polygalae Radix (PR extract; Management No. Q14504) was obtained from Kobayashi Pharmaceutical Co., Ltd. (Ibaraki, Japan). To check the quality of the PR extract, the concentrations of two compounds, i.e., tenuifolin and 3′,6-di-O-sinapoyl sucrose ester, were measured using high performance liquid chromatography (HPLC) analysis according to Hong Kong Chinese Materia Medica Standards. These two compounds are index components, which are used to check the quality of PR, according to the Hong Kong Chinese Materia Medica Standards and China Pharmacopoeia (Chinese Pharmacopoeia Commission, 2015). For the quantification of tenuifolin, the PR extract (0.15 g) was dissolved in 10% sodium hydroxide (30 mL; Cat. No. 198-13765; Wako, Osaka, Japan), and the Solution was heated and refluxed at 110°C for 90 min. The solution was adjusted to pH 4-5 by adding hydrochloric acid (Cat. No. 080-01066; Wako). Water was added to bring the solution up to 100 mL, and 30 mL of the solution was extracted with 1-butanol (50 mL; Cat. No. 026-03326; Wako) twice. The 1-butanol solution was dried, and then resolubilized with methanol (10 mL; Cat. No. 132-06471; Wako). A total of 10 μL of the methanol solution was applied (Shimadzu, Kyoto, Japan) to an HPLC system, on a YMC-Pack ODS-AM AM-312 (6.0 mm i.d. × 150 mm, YMC, Kyoto, Japan) column held at 30°C with a flow rate of 1 mL min-1, and was detected using a photodiode array detector (SPD-M20A, Shimadzu). In the mobile phase, 0.05% phosphoric acid (v/v) (Cat. No. 167-02166; Wako) and acetonitrile (Cat. No. 015-08633; Wako) (33:17) were used. To quantify 3′,6-di-O-sinapoyl sucrose ester, the PR extract (0.3 g) was dissolved in 50% methanol (50 mL). A total of 10 μL of the 50% methanol solution was applied to the HPLC system on a Cosmosil 5C18-AR-II (4.6 mm i.d. × 150 mm, nacalai tesque, Kyoto, Japan) column held at 30°C with a flow rate of 1 mL min-1. Water, acetonitrile, and formic acid (Cat. No. 063-5895; Wako) (860:160:1) were used in the mobile phase. Standard curves were produced to measure concentrations of tenuifolin and 3′,6-di-O-sinapoyl sucrose ester in the PR extract. These two compounds were purchased from TOKIWA phytochemical Co., Ltd. (Sakura, Japan) (tenuifolin, Cat. No. P2515; 3′,6-di-O-sinapoyl sucrose ester, Cat. No. P2939). HPLC profiles of the PR extract are shown in Supplementary Figure S1. Concentrations of tenuifolin and 3′,6-di-O-sinapoyl sucrose ester in the PR extract were 2.89 and 0.86%, respectively.
All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama. The Committee for Animal Care and Use at the Sugitani Campus of the University of Toyama approved all protocols. The approval number for the animal experiments is A2014INM-1 and A2017INM-1, and the confirmation number for the recombinant gene experiments is G2013INM-1.
A novel object recognition test was performed using AD model 5XFAD mice or wild-type (WT) mice (4-month-old, male and female mice; Jackson laboratory, Bar Harbor, ME, United States) as described previously (Kuboyama et al., 2015). Briefly, each mouse was trained to habituate to two identical objects in an open box for 10 min, and were then returned to their home cage. The test session was the performed for 10 min, 22 h later. In the test session, one of the objects was replaced with a novel object in the same place. The number of times the mouse encountered each object was counted. Preference indices were calculated.
After the test session finished, the PR extract (at a dose of 12 or 60 mg kg-1 day-1) or a vehicle solution (sterilized saline) was orally administered, daily, to the mice for 56 days. Saline is an innocuous medium, and treatment with saline is akin to an invasive-free treatment. The day after the last administration, a second trial of the novel object recognition test was performed using different new objects.
After the second trial of the novel object recognition test finished, the mice were euthanized and perfusion-fixed with 4% paraformaldehyde (PFA; Cat. No. 162-16065; Wako) dissolved in phosphate buffered saline (PBS). Brains were cryoprotected in 30% sucrose and frozen on dry ice. Frozen brains were coronally sectioned at 14 μm and further fixed with 4% PFA at 4°C overnight. The sections were then washed with PBS and incubated with blocking solution, i.e., 5% normal goat serum (NGS; Cat. No. 143-06561; Wako) in a solution of 0.3% Triton X-100 (Cat. No. 168-11805; Wako) and PBS for 1 h at room temperature. Then, sections were incubated in a solution containing anti-phosphorylated neurofilament-H (pNF-H) monoclonal antibody (an axonal marker; dilution 1:500; clone SMI-35; Covance, Dedham, MA, United States), anti-Aβ polyclonal antibody (dilution 1:1000; Cat. No. AB5076; Millipore, Billerica, MA, United States), and 1% BSA (Cat. No. 010-25783; Wako) in a solution of 0.3% Triton X-100 and PBS at 4°C overnight. Next, the sections were incubated in a solution containing Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:400; Cat. No. A-11005; Thermo Fisher Scientific, Waltham, MA, United States), Alexa Fluor 488-conjugated goat anti-rabbit IgG (dilution 1:400; Cat. No. A-11008; Thermo Fisher Scientific), and 1% bovine serum albumin (BSA) in a solution of 0.3% Triton X-100 and PBS at room temperature for 2 h. A series of staining procedures was performed using the Shandon Sequenza slide rack (Thermo Fisher Scientific). Fluorescence images (325 μm × 426 μm) were captured using a 20× NA 0.8 dry objective lens (Plan-Apochromat, Carl Zeiss, Oberkochen, Germany) and a charge-coupled device camera (AxioCam MRm, binning set at 1 × 1, Carl Zeiss) on an inverted microscope (AxioObserver Z1, Carl Zeiss). A total of six images were captured for each brain region in each mouse. The density of Aβ plaques in each brain region and densities of degenerated axons merged on the Aβ plaque were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, United States) as described previously (Tohda et al., 2012).
Cerebral cortical neurons (embryonic day 14; ddY mice; SLC, Hamamatsu, Japan) were cultured in 8-well chamber slides (Falcon, Franklin Lakes, NJ, United States) as described previously (Kuboyama et al., 2015). Three days after the culture started, the neurons were treated with either the PR extract (10 and 100 μg mL-1) or a vehicle solution (distilled water) for 30 min. Then, they were treated with either full length Aβ1-42 (1 μM; Sigma, St. Louis, MO, United States) or active partial fragment Aβ25 – 35 (10 μM; Sigma) for 1 h, and fixed with 4% PFA and 4% sucrose in PBS at 37°C for 1 h. Aβ1-42 and Aβ25-35 were aggregated before the treatment as described previously (Kuboyama et al., 2015). The most effective doses of Aβ1-42 and Aβ25-35 were selected based on our previous study (Kuboyama et al., 2015). The entire area in each chamber (7.8 mm × 9 mm) was automatically captured with a 20× dry objective lens (PlanApo λ, Keyence, Osaka, Japan) on an inverted microscope (BZ-710, Keyence). Collapse scores were quantified as described previously (Kuboyama et al., 2015). Briefly, the longest neurite in each neuron was judged as an axon, and growth cones (axonal endings) lacking lamellipodia or possessing fewer than three filopodia were judged as collapsed growth cones (Dotti et al., 1988; Jurney et al., 2002). The collapsed growth cones were scored 1 point, while healthy growth cones were scored 0 point. An average of the scores in each treatment group was shown as a collapse score.
Axonal Growth Assay
After cortical neurons were cultured for 3 days, Aβ25-35 (10 μM) was simultaneously treated with PR extract (at doses of 0.1, 1, 10, and 100 μg mL-1) for 4 days. After that, neurons were fixed with 4% PFA in PBS and blocked with 5% BSA in a solution of 0.3% Triton X-100 and PBS for 1 h at room temperature. The neurons were then incubated in a solution containing anti-pNF-H monoclonal antibody (dilution 1:500), anti-microtubule associated protein 2 polyclonal antibody (a neuronal marker; dilution 1:1000; Cat. No. ab32454; Abcam, Cambridge, United Kingdom), and 5% BSA in a solution of 0.3% Triton X-100 and PBS at 4°C overnight. Next, the neurons were incubated in a solution containing Alexa Fluor 594-conjugated goat anti-mouse IgG (dilution 1:400), Alexa Fluor 488-conjugated goat anti-rabbit IgG (dilution 1:400), DAPI (1 μg mL-1, Sigma), and 3% BSA and 1% NGS in a solution of 0.3% Triton X-100 and PBS for 2 h at room temperature. Fluorescence images (650 μm × 852 μm) were captured using a 10× NA 0.4 dry objective lens on an inverted microscope (AxioObserver Z1). The lengths of axons per neuron were measured using MetaMorph 7.8 software (Molecular Devices, Sunnyvale, CA, United States)…
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