NeuroDefend

NeuroDefend, a traditional Chinese medicine attenuates amyloid-? and tau pathology in experimental Alzheimer’s disease models
Abstract
Alzheimer’s disease (AD) is the most common age related neurodegenerative disorder. Amyloid-? (A?) and hyper-phosphorylated tau accumulation is accountable for the progressive neuronal loss and cognitive impairments usually observed in AD. Currently, medications for AD offers only moderate symptomatic relief but fail to cure the disease and hence development of effective and safe drugs is urgently needed for AD treatment. In this study we investigated a patent-filed Chinese medicine (CM) formulation under the name NeuroDefend (ND) for reducing Amyloid ? (A?) and Tau pathology in the transgenic AD mice models. ND is composed of Rhizoma coptidis, Radix scutellariae, Cortex phellodendri, Radix Salviae Miltiorrhizae (RSM), Rhizoma Corydalis and Uncaria Hook) in 24 formulations with different ratios. Based on the preliminary studies, we selected three formualtions namely ND1, ND15 and ND23 for the in vivo studies. Regular oral administration of NDs (ND1, ND15 and ND23) reversed cognitive dysfunction and memory deficit in 3XTg-AD and 5XFAD mice. In addition NDs significantly reduced insoluble tau protein load and neurofibrillary tangles in 3XTg-AD mice. Furthermore, NDs reduced the levels of CTFs, A?-plaque burden in 3XTg-AD and 5XFAD mice. The selected CM formulations ND1, ND15 and ND23 have the potential to reduce tau and A? pathology and enhance memory function in transgenic mice models. Hence, ND could be a promising candidate for the treatment of AD.

Keywords
NeuroDefend, Alzheimer’s disease, A?-plaque, Neurofibrillary tangles, Chinese medicine
Introduction:
Alzheimer disease (AD) is a common neurodegenerative disorder mostly affecting the aged population and the possibility of complete cure is still uncertain and there is a need of exact curable methods. AD is the most common brain disease in the world which affects 4-8% of the elderly population worldwide. The key hallmark features of AD are: loss of cholinergic neurons; senile plaques (SP) created by accumulation of amyloid ?-peptide (A?) derived from amyloid precursor protein (APP); and tau-associated neurofibrillary tangles (NFTs) (Selkoe and Hardy, 2016).

A? and NFTs are well correlated with cognitive impairment (Yoshiyama et al., 2013). Several critical studies carried out in transgenic mice suggest there is a modulatory link between A? and tau (LaFerla, 2010). Tackling A? and NFTs now seems to be the strategy most likely to succeed. Although SP and NFTs are hallmark symptoms of AD, it is a multifactorial disorder, and is likely to arise from complex genetic and environmental risk factors. Microarray analysis and histopathological studies on SP and NFT-bearing neurons in AD show that behavioural impairments associated with dopaminergic and serotonin dysfunction (markers of psychosis and mood disorders) are seen in AD patients (Geerts et al., 2013, Altar et al., 2009). Although disease-modifying therapies are important, symptomatic therapies addressing cognitive and neuropsychiatric facets of the disease are also very important for the quality of life of patients and caregivers (Geerts et al., 2013, Altar et al., 2009). Currently AD drugs in market are rudimentary and can only relieve certain symptoms but cannot cure the disease. As such, effective and safe drugs are the need for the complete cure of AD.

LaFerla and his group first generated the 3XTg-AD mouse model and are generally used in drug screening and drug discovery. These transgenic mice models express mutated APP, tau proteins (APPswe and TauP301L), and mutated PS1 protein (PS1M146V), which develop both neurofibrillary tangles and amyloid plaques (Oddo et al., 2003b). Interestingly, 3XTg-AD mice exhibited a phenotype of A? and NFTs pathology affecting synaptic plasticity impacting long-term potentiation and memory loss (Pardossi-Piquard et al., 2016). This neuropsychiatric symptom is the most frequent and earliest non-cognitive behavior observed in AD patients (Zhao et al., 2016).

An ideal treatment of AD would be one that combines symptomatic improvement with disease modifying action. Traditional Chinese Medicine (TCM) uses this type of combinational, holistic approach. Traditional Chinese medicine (TCM), the ancient and effective medical system extensively used in East Asia, it has been used for the treatment of multiple neurological diseases including AD. TCM herbs are attracting wide attention for drug discovery and therapeutic approaches. The TCM herbal formula modified HLJDT, our previous work has showed that modified HLJDT (i.e., HLJDT without Huangqin: HLJDT-M, US provisional patent No.61834437) has positive activity compared to the classic formula HLJDT in reducing the amyloid-? (A?) load in both in vitro (Durairajan et al., 2014) and in vivo (Durairajanet al., 2017; Durairajan et al., 2012). Although HLJDT-M shows A?-reducing effect, it does not have a tau-reducing effect and hence, there is a dire need to optimize HLJDT-M for AD treatment.

Based on our preliminary study, developing NeuroDefend (ND) using uniform design to target A? and NFTs would be the novel approach for treating AD; we developed ND by combining HLJDT-M with Danshen, Gouteng and Yanhusuo at a key weight ratio, which turns out to be a potential and effective therapeutic strategy for ameliorating AD.
In this study, we investigated whether ND1, ND23 and ND15 can ameliorate cognitive function and promote the clearance of insoluble tau and A? in 3XTg-AD mice model. Moreover we also evaluated A? pathology in 5XFAD mice model and neuroinflammation in the transgenic mice models.

Materials and methods
2.1. Preparation and quality analysis of NeuroDefend formulation
Initial testing of quality control of individual herb of ND formulation was ascertained according to the Chinese Pharmacopeia by testing the percentage of key marker of each herb. All 6 herbs of three different batches were acquired from the HKBU Chinese Medicine clinic. Dry materials of the plants were grinded into powder using a waring blender. The extraction of ND was done as per the protocol indicated in the Chinese Pharmacopeia. Voucher samples of each herb were deposited in the herbarium of the School of Chinese Medicine. Approximately 1 kg powder of each plant materials according to the ratio of uniform design for ND1, ND23 and ND15 was powdered, extracted in water, steeped in 1L of 70% alcohol overnight and then extracted solutions were filtered. This procedure was repeated twice, for a total of three times. Solutions were pooled, and approximately 12 L of the pooled solution was concentrated by rotary evaporation under vacuum at 50 °C. All the extracts were finally subjected to lyophilization (LABCONCO, Laboratory Construction Company, USA) under vacuum of 105 × 10?3 µbar. Each lyophilized yield was powdered and mixed until homogenous, and then stored at 4° C. Identification and quantitative assessment of reference and active compounds of ND were performed by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) analysis with positive mode and negative mode ESI-MS as described below. In detail, an aliquot of ND1, ND23 and ND15 was dissolved in methanol and centrifuged at 10,000 rpm; the supernatant was filtered and used for analysis. An Agilent 1290 UHPLC system (Agilent Technologies), equipped with an auto sampler, a binary pump and a thermostatic column compartment, was used for chromatographic analysis. The samples were separated on an Acquity UPLC BEH C18 column (1.7um 2.1x100mm) at 40°C. The mobile phase was composed of 0.1% formic acid in water (A) and 0.1% formic acid in ACN The flow rate was 0.1 ml/min. An Agilent 6540 Q-TOF mass spectrometer (Agilent Technologies) equipped with a jet stream electrospray (ESI) ion source was utilized to acquire MS and MS/MS data in positive ion mode. Data acquisition was managed by Mass Hunter B.03 software (Agilent Technologies). Mass spectra were documented for a mass range of 100-1700 m/z for all mass peaks. The injection volume was 2 µl for MS analyses.

2.2. Animals
All animal experiments were approved by the Hong Kong Baptist University Committee on the Use of Human and Animal Subjects in Teaching and Research by the Committee on the Use of Live Animals for Teaching and Research (CULATR #3399-14), at the University of Hong Kong. All animal experiments were performed in accordance with the relevant guidelines and regulations of both HASC and CULATR. We used 5XFAD mice and 3XTg-AD for testing the efficacy of ND.
The “5XFAD” transgenic mice overexpress both mutant human APP(695) with the Swedish (K670N, M671L), Florida (I716V), and London (V717I) Familial Alzheimer’s Disease (FAD) mutations and human PS1 harboring two FAD mutations, M146L and L286V. The 5XFAD transgenic male mice will be crossed with C57 female mice and the resulting transgenic offspring will be used after confirming transgene by PCR according to the supplier’s protocol. 5XFAD mice produce A?42 almost exclusively and it rapidly accumulates to massive cerebral A?42 levels. Amyloid deposition (and gliosis) begins at 2 months and reaches a very large burden at the age of 4 to 5 months in 5XFAD mice with impaired memory in the Morris water test (Oakley et al., 2006; Cho et al., 2014). Mice were housed in a pathogen-free facility under 12 hour light/12 hour dark cycles with food and water provided ad libitum. ND administration started at 2 months of age and lasted for 4 months up to 6 months of age. 3XTg-AD mice show both A? and Tau-associated NFTs, and the amyloid deposition precedes tangle formation, with increased synaptic dysfunction starting from 6 to 12 months of age. In the 3XTg-AD mouse model (Oddo et al., 2003), long-term retention memory deficits in the Morris water maze correlate with intraneuronal A? at 4 months but the NFTs only visible from the 13th month of age. The “3XTg-AD” mouse harbors three human mutant genes, APP with the Swedish mutation, presenilin-1-KI, and TauP301L, on a mixed C57B6/129SVJ background (Oddo et al., 2003). The 3XTgAD mice show various symptoms of AD and unanimously used as the best model for the drug discovery and therapeutic strategies. 3XTg-AD mice and 129svJ were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed in a pathogen-free facility under 12 hour light/12 hour dark cycles with food and water provided ad libitum. ND administration started at 6 months of age and lasted for 8 months up to 14 months of age. The 3XTg-AD mice were fed every day with ND diet admixture with a low dose (1.9 g/kg/d), a middle dose (3.8 g/kg/d), a high dose of herbal extracts (7.6 g/kg/d), or vehicle for 8 months before being killed.

2.3. Open field test:
The open field test was done as described by us previously (Zhang et al., 2014). The locomotor and exploratory behavior of the mice was tested by open field. The apparatus was a square plexiglass box (25×25 cm). The marginal area was selected as being within 10 cm from the walls. Each mouse was positioned at a corner and allowed to move freely; behavioral of activity of each mouse was recorded for 5 min. Behavior parameters such as distance walked, velocity, time spent in movement, and time spent in central/marginal areas were recorded and analyzed by the Ethovision tracking system (Version 3.0, Noldus Information Technology, Leesburg, VA, USA).

2.4. Morris water maze test
The Morris water maze (MWM) test was performed as described by us (Durairajan et al., 2012; Siva et al., 2017). The MWM consists of a circular, 1 m diameter pool made of non-toxic white plastic, filled with water and maintained at 21±1°C. A transparent plexiglass platform of 9 cm diameter and 29 cm height was placed 1 cm below the water surface at a fixed point. Animals were brought to the behavior room, acclimatized and trained. Swim paths were monitored using an automated tracking system (Ethovision XT software). Before all learning and memory tests, visible platform trials were performed to evaluate sensorimotor and/or motivational deficits that could influence performance during the learning task. For the visible platform training (4 trials per day), the platform was placed above the water level with a flag attached, and the platform position was changed for each trial. Hidden-platform training consisted of 6 sessions (1 per day) over 6 days; each session comprised four 60 second trials with a 30 min inter-trial interval. The platform location remained constant in the hidden platform sessions, and the entry point was changed randomly between days. To assess memory retention, a probe trial was conducted after the final hidden-platform training trial by removing the platform and allowing the mice to search for the platform for 60 seconds. The time spent in the target quadrant was taken to indicate the level of memory retention that had taken place after learning. The distance taken to reach the hidden platform and percent time spent in each quadrant of the pool during probe trials of the mouse were recorded and analyzed using an Ethovision video tracking system (Version 3.0, Noldus Information Technology, Leesburg, VA, USA).

2.5. Contextual fear conditioning test
Fear conditioning test was performed in a 30×24×21 cm chamber positioned in a 110×50×60 cm plastic cabinet. In the training session, mice were placed in a chamber and permitted to explore the chamber for 2 min. At the end of 2 min, the audio tone (conditional stimulus: 5 kHz, 70 dB) was given for 28 sec followed by the foot shock (unconditional stimulus: 0.5 mA) from the metal grid on the floor for 2 seconds. Foot shock intensity was determined in a preliminary test on a separate cohort of mice to be the minimal applicable intensity that elicits a response. Each session was last for 30 sec and total experimental time was 5 min. The movements of mice were recorded. Tests that record no movement for more than 1 second was counted as freezing. On day 2, both contextual and cued conditioning tests were performed. In the contextual fear session, mice were returned to the conditioning chamber for 5 minutes without any shock or tone. The time of freezing was recorded and used as an index of contextual memory. After 3 h of rest, the tone-associated, cued conditioning tests were performed. Mice were returned to the chamber, but in a different context. The floor was covered by a white plastic board and a black A-frame contextual plastic insert was placed inside the room. Mice were allowed to explore the chamber for 5 min without any audio tone followed by 5 30-sec audio tones 30 sec apart. Time freezing during and between the audio tones were recorded and used as an index of cued memory (Ugo basile, Italy) (Cheng et al., 2013).

2.6. Tau extraction
The extraction of sarcosyl soluble and insoluble tau species from 3XTg-AD mice brain was prepared as described previously (Myeku et al., 2016). Briefly: For post-nuclear brain lysate preparation, 3XTg-AD mice brain was homogenized in 12 volumes of RIPA buffer with a tablet of a cocktail of protease inhibitors and phosphatase inhibitors (Roche). The homogenates were kept on ice for further processing. The homogenates (2 ml) were centrifuged at 30,000 g for 20 min to separate proteins into soluble (S1, supernatant) and insoluble (pellet). The supernatant containing soluble non-pathological tau proteins was removed. The RIPA-soluble fraction was incubated with 1% sarkosyl for 1 h or 2h at room temperature. After ultra-centrifugation at high speed (100000 g) for 60 min, sarkosyl-insoluble proteins in the pellet fraction were collected. The sarkosyl-soluble protein in supernatant was designated S2 fraction. The pellet was resuspended in 50 µl of Tris-EDTA (10 mM Tris, pH 8.0, and 1 mM EDTA) and labeled P2 (sarkosyl-insoluble tau).

2.7. Western blot analysis
Western blot analysis was performed as described by us (Siva et al., 2017). Cell lysates or brain lysates (equal to 5-10 µg of total protein) were separated on 8% or 10%-15% double layered SDS–PAGE gels and blotted onto PVDF membranes for the detection of PHF-1(phosphorylated tau), AT8, CP13, MC1 and ?-actin (Table1). After blocking with 5% skim milk with 0.1% Tween 20, the blots were probed with primary antibodies overnight at 4 °C with shaking. Blots were washed and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies; goat anti-mouse IgG was used (1:10,000) when the primary antibody was PHF-1(phosphorylated tau), AT8, CP13, MC1, and ?-actin; goat or anti-rabbit IgG were used when the primary antibody was full length APP (FL-APP) and CTFs. After incubation with horseradish peroxidase conjugated with secondary antibody, immunoblots were treated with ECL (Thermoscientific) and developed using X-ray film (Kodak). Films were scanned, and the percentage of band intensity was analysed using Image J software (NIH Image).

2.8. Immunohistochemistry (IHC)
Paraformaldehyde-fixed, frozen brains of 3XTg-AD mice were sectioned in three different regions, around 5-30?m mounted on coated slides, and processed for IHC as described by us (Siva et al., 2017). Three sets of sections in each region were prepared for immunostaining analysis. For tau immunostaining, AT8 (phospho-Tau) was assessed separately. Amyloid deposits in the mice were confirmed with immunostaining using 4G8 amyloid-specific antibodies (Millipore). For staining, 4G8 or AT8 or HT7 or Iba-1 (microgliosis) or GFAP (astrocytosis), antibodies were used to detect the localization of these antigens in the brain slices. Sections through each anatomic region of interest were captured, and a threshold optical density was obtained that discriminates staining from the background. Image analysis was made using Image J analysis (NIH) software. For DAB staining, the brain slices were treated with 1% H2O2 for 10 min to quench the endogenous peroxidase activity. After washed with 0.4% Triton-X100 in PBS, the brain slices were blocked with diluted 2% BSA for 30 min at room temperature. After blocking, the sections were incubated with primary antibody biotinated AT8 and 4G8 for 24 hrs at 4°C. The brain slices were incubated with ABC® Elite (VECTOR, Burlingame, USA) for 30 min at room temperature. Finally the sections were incubated with DAB for 5-10 min. The sections were then air-dried and mounted with Leica mounting medium. The slides were then observed under microscope (Hong Kong, China). Image analysis was made using Image J analysis (NIH) software.

2.9. Statistical analysis
Behavioural data were evaluated with 2-way analysis of variance (ANOVA) for repeated measures with “treatment” and “day” and their interactions as fixed factors. Data was presented as the mean ± S.E.M. Histograms were generated to evaluate the normality of the data. The nonparametric Kruskal-Wallis H test was performed followed by post hoc testing using the Mann-Whitney U test. Data is found to be normally distributed, statistical analysis were performed using parametric one way ANOVA, followed by post hoc comparison of the means using Bonferroni’s or Dunnett’s T3 methods. P<0.05, P<0.01 were considered to be significant. All graphical presentation and statistical tests were executed with GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA).

Results
Herbal preparation and qualitative analysis of NeuroDefend.

We first prepared powder of each plant materials according to the ratio of uniform design for ND1, ND23 and ND15, extracted in water, steeped in 70% alcohol overnight and then extracted solutions were filtered. This procedure was repeated twice, for a total of three times. Solutions were pooled and the pooled solution was concentrated by rotary evaporation under vacuum at 50 °C. All the extracts were finally subjected to lyophilization under vacuum. Each lyophilized yield was powdered and mixed until homogenous, and then stored at 4° C. The whole process is pictured in (Fig. 1A). Qualitative analysis of ND1, ND23 and ND15 by LC-QTOF/MS was carried out at HKBU. The total ion current (TIC) chromatograms corresponding to positive ions of ND1, ND23 and ND15 are shown in (Fig. 1B, 1C, 1D). Then, we compared the LC fingerprints of two different batches of ND1, ND23 and ND15. The base peak chromatograms of the chemical fingerprints from the two batches of ND1, ND23 and ND15 are quite similar, demonstrating that NDs was prepared regularly with good quality (Fig. 1B, 1C, 1D). Thirteen of the most abundant peaks were identified and confirmed by comparison with external marker standards, high-resolution MS and MS/MS fragmentation. The quantitative analysis of the ND1, ND23 and ND15 formulations were assessed and the levels of major and minor bioactive components are given in (Supplementary Fig. 1A and 1B). The major and minor components of NDs are namely berberine, geniposide, palmatine, protopine, tetrahydropalmatine, allocryptopine, coptisine, corynoxeine, corynoxine, isorhyncophyline, salvianolic acid B, Tanshinone 2A, and cryptotanshinone. The ratio of ND1 of 6 herbs Huanglian: Huangbai: ZhiZi: Yanhusuo: Danshen: Gouteng is 0.83: 1.66: 4.16: 2.91: 4.57: 6.24 respectively, the ratio of ND15 of 6 herbs Huanglian: Huangbai: ZhiZi: Yanhusuo: Danshen: Gouteng is 4.15: 3.46: 3.46: 2.76: 6.22: 6.22 respectively and the ratio of ND23 of 6 herbs Huanglian: Huangbai: ZhiZi: Yanhusuo: Danshen: Gouteng is 3.45: 1.92: 1.53: 2.69: 5.76: 4.22 respectively for 1kg lyophilized powder preparation. ND dose in mice was calculated based on the LD50 and the doses are 1.9 g/kg, 3.8 g/kg and 7.6 g/kg.

ND treatment ameliorates spatial learning, memory deficits in 3XTg-AD and 5XFAD mice.

To determine the role of ND1, ND23 and ND15 in 3XTg-AD mice on acquisition of spatial memory and learning. We evaluated the long-term effect of ND1, ND23 and ND15 on the amelioration of cognitive deficits, we used 3XTg-AD mouse model because this model showed the late onset of symptom as similar as human. We orally administered food admixture in 6-month old 3XTg-AD mice with ND1, ND23 and ND15 (1.9 g/kg/day, 3.8 g/kg/day, 7.6 g/kg/day) for 8 months. During the course of treatment ND1, ND23 and ND15 did not influence the animal body weight (Supplementary Fig. 2A, 2B, 2C). Time line for ND treatment and behavior experiments schedule in 3XTg-AD mice as shown in (Fig. 2A). Further to evaluate the long-term effect of ND1, ND23 and ND15 on the amelioration of cognitive deficits in 3XTg-AD mice, we performed Morris water maze experiment. Acquisition of spatial memory, learning and memory retention were evaluated in the ND1, ND23 and ND15 treated 3XTg-AD mice, the acquisition of data during the 6 days of training in the Morris water maze was to analyze the escape latencies of the ND1, ND23 and ND15 treated 3XTg-AD mice during the training period. All groups learned to locate the platform during 6 days of training, as indicated by decreasing escape latencies as training progressed, the escape latencies of ND1, ND23 and ND15 treated groups during 6 day training exhibited significantly shorter travel distance than those of transgenic (Tg) vehicle group (Fig. 2B, 2C, 2D). Further in the acquisition phase of learning, the wild type (WT) groups exhibited significantly shorter travel distance in training than those of Tg placebo group (Supplementary Fig. 3A and 3B). To test the memory retention in ND1, ND23, ND15 treated and Tg- vehicle treatment groups, we executed a probe trial 24?hours after the 6th day training. On the probe trial day ND1, ND23 and ND15 treated group (Fig. 2E, 2F, 2G) spent longer time in probing the platform in the target quadrant than the Tg-vehicle treated group. Further we conclude that memory retention and learning improved in ND1, ND23 and ND15 treatment groups of 3XTg-AD mice compared to the Tg-Vehicle (Fig. 2H).

We then used the open field test to assess and compare the exploratory behavior and locomotor activity of untreated 3XTg-AD mice and those treated with ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day). No significant differences were observed between the vehicle and ND1, ND23 and ND15-treated mice in time spend in Centre, margin and total duration of distance moved (Supplementary Fig. 3C and 3D). ND1, ND23 and ND15 did not cause any notable harmful side effects, so we concluded that ND preparations were well tolerated during the treatment period. These results indicate that all doses of ND1, ND23 and ND15 did not affect the locomotor activity and exploratory behaviors of mice.

Further we investigated Anti-AD effect of ND1 and ND23 in 5XFAD mouse model because this model showed early aggressive A? accumulation. We orally administered ND1 and ND23 as a food admixture in 2-month old 5XFAD mice with ND1 at a concentration of 1.9 g/kg and 3.8 g/kg per day and ND23 at a concentration of 3.8 g/kg and 7.6 g/kg per day until 5-months old. During the course of treatment, ND1 and ND23 did not influence the body weight and is highly tolerable (Supplementary Fig. 2D and 2E). Time line for ND treatment and behavior experiments schedule in 5XFAD mice (Supplementary Fig.2F). At the end of 5 months of age, the hippocampal and amygdala-dependent memory deficit were tested by contextual fear conditioning. ND1 and ND23 ameliorates hippocampal and amygdala-dependent memory deficit in 5XFAD mice, ND1 and ND23 treated animals significantly froze more than vehicle-treated 5XFAD mice in freezing behavior and the auditory cue test (Supplementary Fig. 2G and 2H).

ND treatment mitigates A? pathology, reduces A? plaques in 3XTg-AD and 5XFAD mice.

We further investigated Anti-AD effect of ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day) in vivo to test the long-term effect on the A? reduction; we carried out studies in 3XTg-AD and 5XFAD mouse model. To further confirm the reduction in A?-plaque pathology, we performed immunohistochemistry in the 30 µm brain slices at different regions namely anterior, medial and posterior. ND1, ND23 and ND15 reduced hippocampal A?-plaque burden in brain slices significantly and dose-dependently when compared to the Tg-Vehicle group (Fig. 3A and 3B). These in vivo data suggest that ND1, ND23 and ND15 have the potential to reduce A? pathology and hippocampal A?-plaque burden in a dose dependent manner.
To further confirm we demonstrated Anti-AD effect of ND1 and ND23 in 5XFAD mouse model. To evaluate the A?-plaque pathology, we performed immunohistochemistry in the 30 µm brain slices at different regions namely anterior, medial and posterior. The levels of intra-neuronal A? plaques and the extracellular A? plaques were quantified in amygdala, CA1, CA2, CA3 and cortex regions. ND1 and ND23 reduced hippocampal A?-plaque burden in the 5XFAD mice brain (Supplementary Fig. 4A and 4B). These in vivo data suggest that ND1 and ND23 have the potential to reduce A? plaques in 5XFAD mice model.

ND treatment mitigates Tau pathology and reduces NFTs in 3XTg-AD mice.

To assess tau pathology in the brains of 3XTg-AD mice, we used AT8 monoclonal antibodies to assess the insoluble tau pathology load in the brain by immunohistochemistry. The epitope of AT8 is located outside the region of internal repeats of microtubule binding domains (RT1-4) and requires the phosphorylation of Ser96/Ser404. Immunostaining of AT8-positive neurons in the brains of 3XTg-AD mice revealed the insoluble tau pathology load and neurofibrillary tangles load. To further confirm, we performed immunohistochemistry in the 30 µm brain slices at different regions namely anterior, medial and posterior. ND1, ND23 and ND15 reduce hippocampal neurofibrillary tangles load in brain significantly and dose-dependently (Fig. 4A and 4B) when compared to the Tg-Vehicle group. These in vivo data suggest that ND1, ND23 and ND15 have the potential to reduce tau pathology and hippocampal insoluble tau pathology load and neurofibrillary tangles burden of brain slices in 3XTg-AD mouse model. Altogether, these results demonstrate that ND1, ND23 and ND15 have the potential to reduce tau pathology in 3XTg-AD mouse model.

ND reduces the level of insoluble phosphorylated and misfolded tau in 3XTg-AD mice.

To confirm the above results we performed western blot experiment to prove the above results in protein levels. The insoluble tau in the 3XTg mice brain was separated by the differential extraction and the soluble and insoluble tau fraction from 3XTg-AD mice brain was extracted and designated as sarkosyl-soluble and sarkosyl-insoluble tau, respectively. Phosphorylated tau species in both the soluble and insoluble fraction were detected by AT8, CP13, PHF-1 antibody. The misfolded tau species was detected using MC1 antibody. The phosphorylated and misfolded tau in the sarkosyl-insoluble fraction was significantly reduced in ND1, ND23 and ND15-treated groups when compared to the vehicle-treated group (Fig. 5A-5B, 5C-5D and 5E-5F). However there were no significant differences in the sarkosyl-soluble fraction of ND1, ND23 and ND15-treated groups (1.9, 3.8, 7.6 g/kg/day) when compared to the Tg-vehicle-treated group (Supplementary Fig. 5A, 5B and 5C). Notably, these effects are concomitant with a large reduction of insoluble tau levels suggest that ND1, ND23 and ND15 can reduce abnormal tau aggregation in 3XTg mice.

ND reduces the levels of APP metabolites, A? pathology in 3XTg-AD and 5XFAD mice.

We further investigated Anti-A? effect on APP metabolites in vivo to test the long-term effect of ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day) in 3XTg-AD mouse model. Further we performed the western blot analysis to elucidate the levels of APP metabolites in the brain homogenate, ND1, ND23 and ND15 treatment (1.9, 3.8, 7.6 g/kg/day) significantly and dose-dependently reduced the levels CTFs in brain homogenates (Fig. 6A and 6B). These in vivo data suggest that ND1, ND23 and ND15 have the potential to reduce tau pathology and A? pathology in 3XTg-AD mouse model.

To further confirm the above results, we investigated Anti-A? effect on APP metabolites and assessed the effect of ND1 and ND23 in 5XFAD mouse model. We performed the western blot analysis to elucidate the levels of APP metabolites in the brain homogenate of ND1 and ND23 treated 5XFAD mouse model. ND1 and ND23 treatment (1.9, 3.8, 7.6 g/kg/day) significantly and dose-dependently reduced the levels CTFs in brain homogenates of 5XFAD mice (Supplementary Fig. 6A-6B and 6C-6D). Altogether, these results demonstrate that ND1, ND23 and ND15 have the potential to reduce A? pathology in 3XTg-AD and 5XFAD mouse model.

ND treatment mitigates neuroinflammation in 3XTg-AD and 5XFAD mice.

Further we investigated whether ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day) reduces neuroinflammation in 3XTg-AD mice. As activated microglial cells and reactive astrocytes are very closely associated with neurofibrillary tangles and A? pathology. We evaluated ionized calcium binding adapter molecule 1 (Iba-1) (microgliosis) to identify the condition of neuroinflammation in these ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day) treated 3XTg-AD mice. Immunostaining of Iba-1-positive cells in the brain slice of 3XTg-AD mice revealed that ND1, ND23 and ND15 (1.9, 3.8, 7.6 g/kg/day) treatment decreased reactive Iba-1 positive cell count in dose dependent manner compared to the Tg-vehicle-treated group of 3XTg-AD mice (Fig. 7A and 7B).

To further confirm the above results, we investigated activated microglial cells and reactive astrocytes in the brain slices of 5XFAD mice model. To evaluate the neuroinflammation, we performed immunohistochemistry in the 30 µm brain slices at different regions namely anterior, medial and posterior. As activated microglial cells and reactive astrocytes are very closely associated with neurofibrillary tangles and A? pathology. We demonstrated glial fibrillary acidic protein (GFAP) (astrocytosis) and ionized calcium binding adapter molecule 1 (Iba-1) (microgliosis) to identify the situation of neuroinflammation in these ND1 and ND23 treated 5XFAD mice. Immunostaining of GFAP and Iba-1-positive cells in the brain slices of 5XFAD mice revealed that ND1 and ND23 treatment decreased activated GFAP and reactive Iba-1 positive cell count in dose dependent manner compared to the Tg-vehicle-treated group of 5XFAD mice (Supplementary Fig. 7A and 7B). Putting together, these results demonstrate that ND1, ND23 and ND15 have the potential to reduce neuroinflammation in 3XTg-AD and 5XFAD mouse model.

Discussion
While most of the drugs in the present market either have moderate anti A? effect or anti-tau effect. Here we demonstrate that NeuroDefend could be a promising drug candidate for the treatment of AD because it has both the tau reducing effect and anti A? effect in the 3XTg-AD and 5XFAD mouse model. Until now, the actual therapeutic value of Chinese medicine ND in combination of 6 herbs in terms of anti A? effect, anti-tau effect and cognitive deficits has not been validated. Our study demonstrates for the first time that ND a traditional Chinese medicine formulation in combination of 6 herbs has outstanding and promising therapeutic effect of anti A? effect, anti-tau effect, anti-neuroinflammation and improves memory in 3XTg-AD and 5XFAD mouse model.

In the present study we give evidence that ND a potential Chinese medicine in three formulations with different ratios of herbs namely ND1, ND23 and ND15 improves the acquisition of spatial memory, learning and memory retention in 3XTg-AD and 5XFAD mouse model. Our results are corroborated with our previous studies of HLJDT and HLJDT-M (Siva et al., 2017). Notably ND is more superior and elevated memory improvements in comparison to HLJDT-M. As ND is a combination of HLJDT-M with Danshen, Gouteng and Yanhusuo at a key weight ratio. In addition ND has shown an incredible recovery of memory deficits in both models 3XTg-AD and 5XFAD mice as presented in the results.
Further we evaluated the anti A? effect and anti-tau effect of ND1, ND23 and ND15 in both models 3XTg-AD and 5XFAD mice. We observed a remarkable recovery of transgenic mice models in both aspects of anti A? effect and anti-tau effect on treatment with ND1, ND23 and ND15 in 3XTg mice and ND1; ND23 treatment improved the anti A? effect in 5XFAD mice as shown in the results. In this study we observed ND1, ND23 and ND15 reduced the insoluble tau levels in the brain homogenates of 3XTg-AD mice and decreased AT8 positive neurofibrillary tangles in the brain slices of 3XTg-AD mice. Since a progressive shift of brain tau from soluble tau to insoluble tau plays a mechanistic role in the onset and/or progression of AD (Geerts et al., 2013), NDs facilitated the reduction of insoluble tau and NFTs in mice models may clarify its capability to delay the disease progression of AD. These results are corroborated with our previous studies of HLJDT and HLJDT-M (Siva et al., 2017).
Further we investigated the anti A? effect of ND1, ND23 and ND15 in both models 3XTg-AD and 5XFAD mice. The observed results depicts in aspect of Anti A? pathology in 3XTg-AD mice the brain homogenates showed a remarkable decrease in the CTFs, the APP metabolite which clearly shows the reduction of A? formation in the brain of 3XTg-AD mice model and is corroborated in ND1 and ND23 treated 5XFAD mice models. In Addition we have evaluated the senile plaque and neuritic plaque formation in the brain slices of 3XTg-AD and 5XFAD mice by estimating the 4G8 positive neurons. ND1, ND23 and ND15 treatment significantly reduced the A? plaques in both models 3XTg-AD and 5XFAD mice. These results are corroborated with our previous studies of HLJDT and HLJDT-M (Siva et al., 2017; Durairajan et al., 2012; 2014). Since a progressive shift A?s of brain from soluble form to insoluble form plays a mechanistic role in the onset and progression of AD (Geerts et al., 2013). The ability of NDs to arbitrate a decrease in insoluble A? and plaques may elucidate its capacity to delay the onset of AD in both the mice models.

The NFTs and A? plaques are more associated with reactive astrocytes and activated microglia with the progressive course of AD, due to the augmentation of proinflammatory molecules that mediate the neuronal loss in AD. However we observed that ND1, ND23 and ND15 treatment in 3XTg-AD and 5XFAD mice well reduced the activated microglia and reactive astrocytes, which clearly demonstrates that it mitigates neuroinflammation in both mice models. Nevertheless, because neuroinflammation is a risk factor for neurodegenerative disease, the anti-inflammatory effect of ND1, ND23 and ND15 in 3XTg-AD and 5XFAD mice supports its therapeutic potential for AD. These results are corroborated with our previous studies (Durairajan et al., 2012). Previously we have shown that berberine from HLJDT-M can decrease A?, reactive astrocytes and activated microglia demonstrating anti-inflammatory effect (Durairajan et al., 2012; siva et al., 2017).
In conclusion our results suggest that ND a potential Chinese medicine in three formulations with different ratios of herbs namely ND1, ND23 and ND15 improves the acquisition of spatial memory, learning and memory retention in 3XTg-AD and 5XFAD mice model. ND in a combination of HLJDT-M with Danshen, Gouteng and Yanhusuo at a key weight ratio has shown an outstanding and promising therapeutic effect of anti A? effect, anti-tau effect, anti-neuroinflammation and memory improvement in AD mice model. Our study reveals for the first time that NDs treatment by oral feed admixture reduced tau pathology and A? pathology and enhances memory retention function in 5XFAD and 3XTg-AD mice, respectively. The selected formula ND1, ND23 and ND15 have the potential to reduce tau pathology and A? pathology in mice models. NDs could be a promising therapeutic candidate for the treatment of AD as demonstrated in in vivo studies. Since ND is comprised of several molecules, characterizing the active compounds and understanding the mechanism of action will require further work. ND could be developed as health food supplement or raw materials for medicine to prevent or remedy of AD. This study not only contributed to the disease modification function of ND and also to develop novel TCM candidates at a pharmacology level for the cure of AD. Furthermore, this study will lay a base for the transformation of traditional Chinese medicine and for the development of an anti-AD centered therapeutic strategy on TCM theory.

Acknowledgement
This study was supported by the grants of ITS/253/14, HMRF-12132061 HMRF-13144471 and HMRF-15163481. We thank Prof. Peter Davies (Albert Einstein College of Medicine, Bronx, NY, USA) for his continuous support and providing tau antibodies for the whole research work. We thank Alan Ho for his technical assistance in the LCMS analysis of the herbal extracts and their pure compounds. We thank Prof. J.D. Huang and Prof. Sookja K. Chung for providing the behavioral facility for the whole research work. We would like to thank Dr.Martha Dahlen for her English editing on this manuscript.
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