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Sodium oligomannate therapeutically remodels gut microbiota and suppresses gut bacterial amino acids-shaped neuroinflammation to inhibit Alzheimer’s disease progression

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Recently, increasing evidence has suggested the association between gut dysbiosis and Alzheimer’s disease (AD) progression, yet the role of gut microbiota in AD pathogenesis remains obscure. Herein, we provide a potential mechanistic link between gut microbiota dysbiosis and neuroinflammation in AD progression. Using AD mouse models, we discovered that, during AD progression, the alteration of gut microbiota composition leads to the peripheral accumulation of phenylalanine and isoleucine, which stimulates the differentiation and proliferation of pro-inflammatory T helper 1 (Th1) cells. The brain-infiltrated peripheral Th1 immune cells are associated with the M1 microglia activation, contributing to AD-associated neuroinflammation. Importantly, the elevation of phenylalanine and isoleucine concentrations and the increase of Th1 cell frequency in the blood were also observed in two small independent cohorts of patients with mild cognitive impairment (MCI) due to AD. Furthermore, GV-971, a sodium oligomannate that has demonstrated solid and consistent cognition improvement in a phase 3 clinical trial in China, suppresses gut dysbiosis and the associated phenylalanine/isoleucine accumulation, harnesses neuroinflammation and reverses the cognition impairment. Together, our findings highlight the role of gut dysbiosis-promoted neuroinflammation in AD progression and suggest a novel strategy for AD therapy by remodelling the gut microbiota.
GV-971 inhibits neuroinflammation by harnessing amino acid metabolism. a Pathway enrichment analysis of faecal metabolites in 7-month-old 5XFAD (Tg) mice with or without GV-971 treatment (100 mpk) using MBROLE (n = 6-8). A partial ist of the enrichment results is presented with KEGG modules and KEGG enzyme interactions which have been screened using a criterion of FDR-adjusted P-value < 0.05. b Lists of the most important blood amino acids of the random forest model ranked from most to least important between WT (2 m-9 m) and Tg (2 m-9 m) group from a ROC curve analysis. Red indicated high concentration, green indicated low concentration. (n = 30 for WT, n = 26 for Tg). c Changes in histidine, phenylalanine and isoleucine levels in the feces of WT, 5XFAD mice (Tg), and GV-971-treated Tg mice (100 mpk) (n = 6-11) at 7-month old. Colours in the heatmap indicate relative metabolite levels; red indicates metabolites that are upregulated, and blue indicates metabolites that are downregulated. d Changes in histidine, phenylalanine and isoleucine levels in the blood of WT, 5XFAD mice (Tg), and GV-971-treated Tg mice (100 mpk) (n = 6-7) at 7-month old. Colours in the heatmap indicate relative metabolite levels. Red indicates metabolites that are upregulated, and blue indicates metabolites that are downregulated. e The effects of GV-971 on the differentiation of naïve CD4 + T cells (Th0 cells) to Th1 cells induced by phenylalanine and isoleucine in vitro. Naïve CD4 + T cells were cultured for 5 days with/ without GV-971 in the presence of phenylalanine (1 mM) or isoleucine (1 mM). The frequency of Th1 (CD4 + IFN-γ + ) cells was tested by flow cytometry (see Materials and methods). GV-971 was used at a final concentration of 100 µg/mL. The data are presented as mean ± standard error of the mean (mean ± SEM); n = 3 replicates per group, one of three replicated results was represented. Left, *P < 0.05, **P < 0.01 by oneway ANOVA (F (2, 6) = 15.64). Right, *P < 0.05, **P < 0.01 by one-way ANOVA (F (2, 6) = 10.35). f The effects of GV-971 on the proliferation of Th1 cells induced by phenylalanine and isoleucine. Th1 cells were stained with CellTrace and cultured for 4 days with/without GV-971 in the presence of phenylalanine (1 mM) and isoleucine (1 mM). The density of CellTrace fluorescence in Th1 (CD4 + IFN-γ + ) cells was tested by flow cytometry (see Materials and methods). GV-971 was used at a final concentration of 100 µg/mL. The data are presented as mean ± standard error of the mean (mean ± SEM), n = 3 replicates per group, one of three replicated results was represented. *P < 0.05, ***P < 0.001 by one-way ANOVA (F (4, 9) = 28.34). Phe, phenylalanine; Ile, isoleucine. g Frequency of blood Th1 cell changes in C57 mice after 4-day intraperitoneal (i.p.) injection of phenylalanine and isoleucine (n = 8). ***P < 0.001 by one-way ANOVA (F (2, 21) = 101.8). h Random forest classification of amino acid changes in healthy controls (HC) and mild cognitive impairment (MCI) due to AD patients. The amino acids are ranked by mean decrease in classification accuracy (first cohort, n = 9 for MCI due to AD, n = 18 for HC). Red indicated high concentration, green indicated low concentration. i Frequency of Th1 cells in the blood of healthy controls (HC) and mild cognitive impairment (MCI) due to AD patients (first cohort, n = 8 for MCI due to AD, n = 9 for HC). *P < 0.05 by Student's t-test. j Levels of phenylalanine and isoleucine in the blood of healthy controls (HC) and mild cognitive impairment (MCI) due to AD patients (second cohort, n = 22 for both groups). *P < 0.05 by Student's t-test
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ARTICLE OPEN
Sodium oligomannate therapeutically remodels gut microbiota
and suppresses gut bacterial amino acids-shaped
neuroinammation to inhibit Alzheimers disease progression
Xinyi Wang
1
, Guangqiang Sun
1
, Teng Feng
1
, Jing Zhang
1
, Xun Huang
2
, Tao Wang
3
, Zuoquan Xie
2
, Xingkun Chu
1
, Jun Yang
1
,
Huan Wang
2
, Shuaishuai Chang
1
, Yanxue Gong
1
, Lingfei Ruan
1
, Guanqun Zhang
1
, Siyuan Yan
1
, Wen Lian
1
, Chen Du
1
, Dabing Yang
1
,
Qingli Zhang
4
, Feifei Lin
4
, Jia Liu
4
, Haiyan Zhang
2
, Changrong Ge
1
, Shifu Xiao
3
, Jian Ding
2
and Meiyu Geng
2
Recently, increasing evidence has suggested the association between gut dysbiosis and Alzheimers disease (AD) progression, yet
the role of gut microbiota in AD pathogenesis remains obscure. Herein, we provide a potential mechanistic link between gut
microbiota dysbiosis and neuroinammation in AD progression. Using AD mouse models, we discovered that, during AD
progression, the alteration of gut microbiota composition leads to the peripheral accumulation of phenylalanine and isoleucine,
which stimulates the differentiation and proliferation of pro-inammatory T helper 1 (Th1) cells. The brain-inltrated peripheral Th1
immune cells are associated with the M1 microglia activation, contributing to AD-associated neuroinammation. Importantly, the
elevation of phenylalanine and isoleucine concentrations and the increase of Th1 cell frequency in the blood were also observed in
two small independent cohorts of patients with mild cognitive impairment (MCI) due to AD. Furthermore, GV-971, a sodium
oligomannate that has demonstrated solid and consistent cognition improvement in a phase 3 clinical trial in China, suppresses gut
dysbiosis and the associated phenylalanine/isoleucine accumulation, harnesses neuroinammation and reverses the cognition
impairment. Together, our ndings highlight the role of gut dysbiosis-promoted neuroinammation in AD progression and suggest
a novel strategy for AD therapy by remodelling the gut microbiota.
Cell Research (2019) 0:117; https://doi.org/10.1038/s41422-019-0216-x
INTRODUCTION
Despite the tremendous efforts made in the treatment of
Alzheimers disease (AD), the past decades have witnessed the
continuous failure of β-amyloid (Aβ)- or tau-centric therapeutic
strategies in late-stage clinical trials, which impels the reconsi-
deration of new therapeutic strategy for this complicated
disease.
1,2
Recently, a plethora of studies have shown the dynamic
interaction between the intestinal microbiota and host innate and
adaptive immune system.
3,4
The dysbiosis of gut microbiota could
jeopardize host immune responses and promote the development
of various inammatory disorders.
5
This also appears to be the
case for AD-associated inammation. Emerging evidence from
both animal and human studies supports the association between
dysbiosis of the gut microbiota and the microglia activation
during AD development.
68
For example, Minter et al. discovered
that perturbations in gut microbial diversity inuenced neuro-
inammation and amyloidosis. Antibiotics-treated mice showed a
signicant decrease in plaque-localized microglial activation
positive for IBA-1.
9
Besides, gut microbiota-produced lipopolysac-
charide (LPS) was found in the post-mortem brain samples of AD
patients,
10
and peripheral injection of LPS could promote
microglial activation.
11,12
All the evidence suggests that gut
microbiota is likely involved in regulating microglia activation and
neuroinammation in AD.
In addition to the microglia activation, the role of inltrating
peripheral immune cells, such as CD4
+
and CD8
+
T cells, etc., in
AD-associated neuroinammation is increasingly appreciated. For
example, peripheral type 1 and type 17 T-helper (Th1, Th17) cells
have been reported to be associated with releasing of inamma-
tory cytokines in multiple AD mouse models.
13,14
Consistent with
these evidences, peripheral inltrated lymphocytes were observed
in the brain of both transgenic mouse models and AD patients.
13
Additionally, in the post-mortem brains of AD patients, both CD4
+
and CD8
+
T cells were detected.
15
Current understanding of the mechanistic link between the gut
and brain in AD progression is still very limited.
16,17
It remains
unknown which types of inltrated immune cells are functionally
involved in AD development.
18,19
The underlying driving force
that promotes peripheral immune cells to inltrate the brain,
leading to the enhancement of the residential neuroinammation,
Received: 14 April 2019 Accepted: 29 July 2019
1
Shanghai Green Valley Pharmaceutical Co., Ltd, Shanghai 201203, China;
2
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, China;
3
Department of Geriatric Psychiatry, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine; Alzheimers Disease
and Related Disorders Center, Shanghai Jiao Tong University, Shanghai 200025, China and
4
Institutional Technology Service Center, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
Correspondence: Meiyu Geng (mygeng@simm.ac.cn)
These authors contributed equally: Xun Huang, Tao Wang, Zuoquan Xie
These authors contributed equally as co-rst authors: Xinyi Wang, Guangqiang Sun, Teng Feng, Jing Zhang
www.nature.com/cr
www.cell-research.com
©The Author(s) 2019
remains obscure. Moreover, as the importance of microbiota
metabolites is starting to unveil, it will be imperative to
comprehend the specic metabolites that are involved in linking
gut microbiota and brain neuroinammation in AD progression.
This study aims to investigate the mechanistic linkage between
gut microbiota and AD progression, and to explore the potential
intervention strategies. To this end, we investigated the associa-
tion between dynamic changes of gut microbiota and neuroin-
ammation at different stages of AD development, and probed
the possible mechanistic links between intestinal microbiota and
neuroinammation in the aspects of metabolites. In particular,
we took the advantage of an oligosaccharide anti-AD drug,
designated as GV-971, as a probe to assess the potential
intervention approach by targeting the gut microbiota. GV-971
is a mixture of acidic linear oligosaccharides ranging from dimers
to decamers (molecular weight up to ~1 kDa). Recently, GV-971
has completed a Phase 3 clinical trial for AD in China and
successfully met its primary endpoint in improving cognition
impairment (unpublished data).
We have revealed that gut microbiota patterns and amino acid-
derived metabolites are important for the inltration of specic
types of immune cells, which drives neuroinammation during AD
progression. GV-971 therapeutically harnesses the abnormal
production of amino acids, inltration of immune cells to the
brain, and in turn neuroinammation via remodelling the gut
microbiota. The elucidation of the mechanisms underlying the
neuroimmune regulation by microbiota may hold promise for
developing novel therapeutics for AD patients.
RESULTS
AD progression is associated with the alteration of gut microbiota
and immune cell inltration
To assess the role of gut microbiota alteration in AD pathogenesis,
we used the 5XFAD transgenic (Tg) mouse model, which is widely
used in AD study for faithfully recapitulating AD-associated
pathological features including the severely accelerated cognitive
impairment, amyloid deposition in the 2
nd
postnatal month,
synaptic degeneration in the 4
th
postnatal month, and behavioural
changes in the 6
th
month.
2023
Consistent with these reports,
2427
we observed rapid accumulation of Aβplaque deposition in the
cortex and hippocampus beginning from the 3
rd
postnatal month
in Tg mice compared to the age-paired wild-type (WT) mice
(Supplementary information, Fig. S1a). Tau phosphorylation in the
brain of Tg mice was rst found in the 5
th
month and increased
gradually compared to the age-paired WT mice (Supplementary
information, Fig. S1b). The synaptophysin expression level in the
hippocampus signicantly decreased from the 7
th
to the 9
th
months, indicative of synaptic degeneration (Fig. 1a), while the
behavioural test in Tg mice showed a signicant decline in
discrimination learning at 9 months of age (Fig. 1b).
We then compared the enterotypes of Tg mice and WT mice at
different stages in AD progression using 16S ribosomal RNA (rRNA)
gene amplicon sequencing. The sequences were grouped into
provisional clusters as operational taxonomic units (OTUs).
Enterotype analysis revealed a notable clustering effect in the
gut microbiome, with Tg mice clustered into a norank genus
under the family of Muribaculaceae and WT mice clustered into
the Lactobacillus genus (Fig. 1c). Using principal component
analysis (PCA), which simplies the complexity in high-
dimensional data while retaining trends and patterns upon a
series of time points (also see Materials and methods
28
), we
revealed a remarkable shift in the gut microbiota composition
during AD progression in Tg mice. Hardly any changes were
observed in WT mice (Fig. 1d; Supplementary information,
Fig. S1c). Using OTUs to track the dynamics of the abundance of
different bacterial phyla in Tg mice, we found two distinct changes
of gut microbiota proles in Tg mice during disease progression.
At 2 months of age, Bacteroides, Firmicutes and Verrucomicrobia
were the three most abundant bacteria at the phylum level
(47.3%, 33.0% and 12.2%, respectively). At 7 months of age,
Firmicutes became the predominant phylum (62.8%), while the
abundance of Bacteroidetes and Verrucomicrobia markedly
decreased, indicating an alteration of the types of bacteria
(Fig. 1e). Representative bacteria from Tg mice at each time point
were further explored (Supplementary information, Fig. S1d, e and
Table S1). These results indicated that the gut microbiota of Tg
mice was highly dynamic, in great contrast to that of the WT mice.
Similar results were also obtained in the APP/PS1 double
transgenic mouse model, another widely used model for AD
study (Supplementary information, Fig. S1f, g).
20,2931
Previous studies have suggested that gut microbiota is involved
in triggering neuroinammation in the brain.
18,32
We hypothe-
sized that the observed gut microbiota alteration during AD
progression might be associated with neuroinammation. To test
this hypothesis, we evaluated the inammatory status of Tg mice.
Immunostaining of IBA1, a hallmark of microglial activation, in AD
mouse brain sections revealed two evident stages of microglial
activation, at the 3
rd
month and the 79
th
months, respectively
(Fig. 1f; Supplementary information, Fig. S1h). Given that
microglial activation can be categorized into two opposite types,
the pro-inammatory M1 and the neuroprotective M2 subtype,
33
we also carefully characterized M1 and M2 phenotypes. At early
stage of 2-3-month old, both M1 and M2 microglia increased,
while in the following months, M1 subtype continued to increase
and peaked at 7-9 months, whereas M2 type microglia declined
from 3 to 5 month and maintained a low level thereafter (Fig. 1g).
In addition to microglia activation, AD-associated neuroinam-
mation is known to involve the inltration of peripheral immune
cells.
3436
We therefore also analyzed the inltrating peripheral
immune cells in the brain during AD progression. It was observed
that the frequency of CD45
high
cells in the brain was signicantly
higher in Tg mice than that in WT mice, similar to the result of
IBA1 staining (Fig. 1f, h). We further proled changes of CD45
high
cell subtypes at a series of time points during AD progression
(Fig. 1i). Using the k-means clustering (see Materials and methods),
we revealed that the alteration of CD4
+
T cells, as the major
proportion of CD45
high
cells, closely recapitulated the change of
M1 cells (Fig. 1i, j). Inltrating Th1 and Th2 cells, two major
subtypes of CD4
+
cells, exhibited similar dynamics to that of M1
and M2 microglial cells over the period that we examined (Fig. 1k).
It appeared to us that, as the pattern of the gut microbiota shifted,
the immune cell population tended to change to a Th1- and M1-
dominated state. Similar results were also found in the APP/PS1
model (Supplementary information, Fig. S1i, j) and other AD
mouse models by querying public databases (Supplementary
information, Fig. S2).
37,38
We also analyzed the correlation between gut microbiota
abundance and brain immune cell frequency at both early and
late stages of AD progression. We noted that the abundance
changes of bacteria were aligned with the alterations of Th2 and
M2 cells at the early stage (23 months) (Fig. 1l, top), but
correlated with those of Th1 and M1 cells at the late stage
(79 months) (Fig. 1l, bottom). The bacteria that were signicantly
interrelated with immune cells were listed in the right panel of
Fig. 1l (list of the full names is in Supplementary information,
Table S2). Overall, these results indicated that gut bacteria were
associated with the inltration of peripheral immune cells and
neuroinammation occurrence during AD progression.
Gut microbiota is required for immune cell inltration and
microglial activation in the brain
To determine whether the gut microbiota change is required for
driving peripheral immune cell inltration and in turn neuroin-
ammation in AD progression, we used an antibiotic cocktail
containing ampicillin (0.1 mg/mL), streptomycin (0.5 mg/mL), and
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Cell Research (2019) 0:1 – 17
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colistin (0.1 mg/mL) to ablate gut microbiota. Antibiotic treatment
in Tg mice resulted in a marked reduction in microbial abundance
in the gut (Fig. 2a; Supplementary information, Table S3). Along
with this change, we observed a reduction in both inltrating pro-
inammatory Th1 cells (Fig. 2b) and M1 cells (Fig. 2c) in the brain.
Further, these ndings were conrmed by a co-housing
experiment, in which WT mice were co-housed with Tg mice of
the same age for 7 months since birth. The co-housed WT mice
displayed the decline in discrimination learning, comparable to
that of Tg mice, indicating that the long-term exposure to the
Tg bacteria could cause cognitive impairment (Supplementary
information, Fig. S3a). Analysis of the gut microbiota change in
these mice indicated that the composition of the gut microbiota
was quite similar between co-housed WT mice and Tg mice, but
signicantly different from that of WT mice of the same age under
separate housing (Fig. 2d). Moreover, inltrating Th1 cells between
co-housed WT and Tg mice were comparable as well, which were
both signicantly higher than that of separately housed WT mice
(Fig. 2e). Meanwhile, M1 cells were only slightly increased in the
co-housed WT mice (Fig. 2f), suggesting that microbiota alteration
mainly inuenced the inltrating immune cells rather than the
resident immune cells. In line with the immune cell change,
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Cell Research (2019) 0:1 – 17
cytokine expression in the brain showed a marked similarity
between co-housed WT and Tg mice, but distinct from those of
WT mice (Fig. 2g; Supplementary information, Table S4).
We also performed faecal microbiota transplantation (FMT)
experiments using C57BL/6 WT mice. For C57 WT mice with
hippocampal Aβinjection, oral administration of faecal microbiota
from 7-month Tg mice resulted in a marked increase in Th1 cells
and a reduction in Th2 cells in the brain (Supplementary
information, Fig. S3b). Reversely, transplantation of WT feces
decreased brain Th1 cells in the recipient Tg mice (Supplementary
information, Fig. S3c). Together, these ndings suggest that the
gut microbiota alteration drives peripheral immune cell inltration
and neuroinammatory activation in AD progression.
GV-971 exhibits ameliorative effects on cognitive impairment
The essential role of gut microbiota in AD progression revealed
herein may suggest the therapeutic implications by the interven-
tion of gut microbiota. To test this hypothesis, we took advantage
of GV-971 (Fig. 3a). In 9-month-old APP/PS1 mice treated with GV-
971 for three months until 13-month old, GV-971 exhibited
signicant ameliorative effect on the cognitive impairment, as
shown by the enhanced spatial learning and memory perfor-
mance of APP/PS1 mice in both training trial (Fig. 3b) and probe
trial (Fig. 3c) in the Morris Water Maze (MWM) task. GV-971 also
signicantly improved the mice performance in Y maze (Fig. 3d).
Recently, GV-971 has shown the therapeutic effect on reversing
cognition impairment in AD patients in a 36-week multi-center,
randomized, double-blind, placebo-controlled Phase 3 clinical trial
in China. Given that carbohydrates, in the forms of monosacchar-
ide or oligosaccharide, are the primary nutrient source for bacteria
and have shown diverse modulatory effects on bacteria,
3942
we
were interested to explore whether GV-971 could affect the gut
microbiota.
GV-971 alleviates neuroinammation by shaping the gut
microbiota
Intriguingly, one-month oral administration of GV-971 in Tg mice
beginning from 7-month-old age markedly altered the composi-
tion of gut microbiota in a bi-directional way (Fig. 4a, b;
Supplementary information, Fig. S4a and Table S2). In line with the
gut microbiota alteration, GV-971 treatment in Tg mice disrupted
the correlation previously seen between brain lymphocytes and
gut bacterial change (Fig. 4c; Supplementary information, Fig. S4b,
c and Table S2), decreased brain Th1 cells (Fig. 4d), signicantly
reduced microglial activation (Fig. 4e), and decreased multiple
brain cytokines levels (Fig. 4f; Supplementary information,
Table S4). In parallel, GV-971 treatment signicantly reduced the
Aβplaque deposition, tau phosphorylation, and ameliorated the
decline in discrimination learning in Tg mice (Fig. 4gi; Supple-
mentary information, Fig. S4d).
Further, we did FMT, transplanting feces of GV-971-treated
Tg mice to the recipient C57BL/6 WT mice which were
Fig. 1 Gut dysbiosis and immune cell changes during disease progression in 5XFAD transgenic (Tg) mice. aChanges in the relative RNA
expression levels of synaptophysin in the hippocampus of 5XFAD transgenic (Tg) mice at 2, 3, 5, 7 and 9 months and in wild-type (WT) mice at
2 months (n=512). The data are presented as mean ± standard error of the mean (mean ± SEM) relative to the expression level of actin. *P<
0.05, **P<0.01 by one-way ANOVA (F (5, 43) =2.952). bChanges in the time out of 10
4
s taken to achieve 80% success (see Materials and
methods) in a test to evaluate the discrimination learning abilities of 5XFAD transgenic (Tg) mice at 2, 3, 5, 7, and 9 months and wild-type
(WT ) mice at 2 months (n=48). Data are presented as mean ± standard error of the mean (mean ± SEM). *P<0.05 by Students t-test. s
seconds. cEnterotype analysis at the genus level of the gut microbiomes of 5XFAD transgenic (Tg) mice and wild-type (WT ) mice at 2-, 3-, 5-,
7- and 9-month old (n=410). The separation of gut microbial taxa into a norank genus under the family of Muribaculaceae of Tg mice and the
genus Lactobacillus enterotypes of WT mice is achieved by calculating Bray-Curtis distance based on the relative abundance at the genus level
and clustered using PAM (Partitioning Around Medoids). The data are most naturally separated into two clusters, as determined by the
Calinski-Harabasz (CH) index and represented using principal coordinate analysis (PCoA). The shapes and colours of the points indicate
samples from each individual from various months. The coloured ellipses indicate the 0.95 condence interval (CI) ranges within each
enterotype group. M months. dPrincipal component analysis (PCA) of the gut microbiome composition of WT and 5XFAD transgenic (Tg)
mice on the operational taxonomic unit (OTU) level at different time points (n=410). The shapes and colours of the points indicate samples
from each individual from various months. The coloured ellipses indicate 0.95 condence interval (CI) ranges within each tested group. M
months. eAbundance changes of operational taxonomic units (OTUs) in the overall population in the gut microbiome of 5XFAD transgenic
(Tg) mice at various months, coloured at the phylum level on a stream graph (n=410). The two most abundant phyla, Bacteroidetes and
Firmicutes, are labelled on the graph. Colours indicate different phyla of the gut microbiota. fChanges in the positive densities of IBA1
immune-uorescent staining, reecting activation of microglial cells in the hippocampus of 5XFAD transgenic (Tg) mice at 2, 3, 5, 7, and
9 months relative to the values of 2-month-old wild-type ( WT ) mice (n=27). The data are presented as mean ± standard error of the mean
(mean ± SEM); lines are tted with a cubic spline. gChanges in activated M1 and M2 type microglia detected in the whole-brain homogenates
of 5XFAD transgenic (Tg) mice at 2, 3, 5, 7 and 9 months (n=48). M1-type microglia (CD45
low
CD11b
+
CX3CR1
+
Siglec-H
+
CD86
+
) and M2-type
(CD45
low
CD11b
+
CX3CR1
+
Siglec-H
+
CD206
+
) microglia were detected by ow cytometry, and their cell counts are presented relative to the
frequency of CD45
low
CD11b
+
cells. Red points and lines: M1 microglia. Green points and lines: M2 microglia. The data are presented as mean
± standard error of the mean (mean ± SEM); lines are tted with a cubic spline algorithm. hChanges in inltrating cells (CD45
high
) detected in
the whole-brain homogenates of 5XFAD transgenic (Tg) mice (red points and lines) and WT mice (black points and lines) at different time
points as detected by ow cytometry (n=48). Cell counts are presented relative to the frequency of CD45
+
cells and formatted as mean ±
standard error of the mean (mean ± SEM). Lines are tted with a cubic spline algorithm. iChanges in CD45
high
cells in 5XFAD transgenic (Tg)
mice at different time points (n=48). On the barplot, cell counts are presented relative to the frequency of CD45
high
cells. Colours indicate
different subtypes of CD45
high
cells: Neu, neutrophils; DC, dendritic cells; NK, natural killer cells; Mo/Mϕːmonocytes and macrophages; B, B
cells; Others, unclassied cells. jChanges in inltrating CD4 T cells (CD45
high
CD4
+
) detected in the whole-brain homogenates of 5XFAD
transgenic (Tg) mice (red points and lines) at 2, 3, 5, 7 and 9 months as detected by ow cytometry (n=48). Cell counts are presented
relative to the frequency of CD45
high
cells and formatted as mean ± standard error of the mean (mean ± SEM). Lines are tted with a cubic
spline. kChanges in inltrating peripheral Th1 and Th2 cells detected in the whole-brain homogenates of 5XFAD transgenic (Tg) mice at 2, 3,
5, 7 and 9 months (n=48). Th1 cells (CD45
high
CD4
+
CXCR3
+
) and Th2 cells (CD45
high
CD4
+
CCR4
+
) were detected by ow cytometry, and
presented relative to the frequency of CD45
high
CD4
+
T cells. Red points and lines: Th1 cells. Green points and lines: Th2 cells. The data are
presented as mean ± standard error of the mean (mean ± SEM). Lines are tted with a cubic spline. lCorrelation between the trend changes of
the frequency of brain lymphocytes and the abundance of gut microbiota represented at genus level during the Th2/M2-related stage and
Th1/M1-related stage in the early and late phase (23 months and 79 months), respectively (left panels, n=48). Bacteria that are
signicantly correlated with Th2/M2 or Th1/M1 in 5XFAD mice are listed in the right-hand panels. Squares in red (positive correlation) or blue
(negative correlation) with a yellow asterisk (*) indicate signicant correlations with P-values < 0.05 measured by the Pearson parametric
correlation test (see Materials and methods). f family, M months, T regulatory cells Treg, Mo monocytes, B B cells, DC dendritic cells, NK
natural killer cells
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Fig. 2 The gut microbiota is required for immune cell inltration and microglial activation. aThe effects of ve-month oral gavage of
antibiotics on the relative abundance of gut microbes in 7-month-old 5XFAD transgenic (Tg) mice (n=57). ABX, a cocktail of mixed
antibiotics composed of ampicillin (0.1 mg/mL), streptomycin (0.5 mg/mL) and colistin (0.1 mg/mL). Different genera of gut microbes are
coloured differently, and their changes in relative abundance are presented on the barplot. The abundance of Bacteroides in Tg mice is below
0.01 and was thus not shown in both groups. b-c The effects of ve-month oral gavage of antibiotics on the frequency of Th1 cells (b) and M1-
type microglia (c) in the brain homogenate of 7-month-old 5XFAD transgenic (Tg) mice. Cell counts of Th1 cells (CD45
high
CD4
+
CXCR3
+
) are
presented relative to the frequency of CD45
high
cells (b), while those of M1-type microglia (CD45
low
CD11b
+
CX3CR1
+
Siglec-H
+
CD86
+
) are
presented relative to the frequency of CD45
low
cells (c). Both are detected by ow cytometry, n=7, the data are presented as mean ± standard
error of the mean (mean ± SEM). dThe relative abundance of gut microbes at the genus level in WT, co-housed WT and 5XFAD transgenic ( Tg)
mice (n=67). All three groups of mice were at 7-month old. Different colours represent different genera. Co-housed WT: WT mice that were
housed with Tg mice. e-f Changes in the frequency of Th1 cells (e) and M1 type microglia (f) in the brain homogenates of 7-month-old WT, co-
housed WT and 5XFAD transgenic (Tg) mice (n=47). Th1 cells (CD45
high
CD4
+
CXCR3
+
) are presented relative to the frequency of CD45
high
cells (e), while the frequency of M1-type microglia (CD45
low
CD11b
+
CX3CR1
+
Siglec-H
+
CD86
+
) are presented relative to the frequency
of CD45
low
CD11b
+
cells (f). Both are detected by ow cytometry. The data are presented as mean ± standard error of the mean (mean ± SEM).
*P< 0.05, **P<0.01 by Students t-test. gLevels of cytokine proteins in the brain homogenates of WT, co-housed WT and 5XFAD transgenic
(Tg) mice at 7-month old as detected by a cytokine antibody array (n=56). Colours in the heatmap indicate relative cytokine levels; red
indicates cytokines that are upregulated, and blue indicates cytokines that are downregulated
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Cell Research (2019) 0:1 – 17
intraventricularly injected with aggregated Aβ. Feces of GV-971-
treated Tg mice resulted in decreased Th1 cells in the brain of
recipient mice as compared to that of Tg mice without GV-971
treatment (Supplementary information, Fig. S4e). Consistently,
antibiotic treatment impaired the effect of GV-971 on gut
microbiota, Th1 cells, IBA1 levels and cytokine expression in the
APP/PS1 mouse model (Supplementary information, Fig. S4f-j). In
addition, treatment with oligo-guluronic acid (PG), an epimer of
GV-971, or with polymannuronate sulfate, polyguluronate sulfate,
heparin, heparan sulfate did not show the therapeutic effects
(data not shown), indicating that the sugar backbone of GV-971
may account for its specic impact on gut microbiota. All these
data suggested that GV-971 could alleviate neuroinammation
and cognition decline via modulating gut dysbiosis.
GV-971 inhibits neuroinammation by regulating amino acid
metabolism
Emerging evidence has highlighted the role of gut microbiota-
associated metabolites in inuencing the host immune system.
43
To test the possible involvement of metabolites in immune
modulation, the supernatant of in vitro-cultured feces from 7-
month-old Tg mice
4446
was added to naïve CD4
+
T cell culture
(see Materials and methods), which stimulated the differentiation
of naïve CD4
+
T cells to Th1 and Th2 cells. In contrast, fecal
supernatant of Tg mice treated with GV-971 inhibited Th1
differentiation and promoted Th2 differentiation (Supplementary
information, Fig. S5a).
We next employed a non-targeted metabolomics technique to
characterize the fecal metabolome. A total of 11289 metabolites
were identied in faecal samples from WT, Tg and GV-971-treated
Tg mice (Supplementary information, Fig. S5b-c). Among those
metabolites matched to database (Supplementary information,
Fig. S5d-e), the abundance of 124 metabolites, as annotated by
METLIN database, was signicantly changed in Tg mice as
compared to that in WT mice (Supplementary information, Fig. S5f
and Table S5). These altered metabolites were further annotated
with the Human Metabolome Database (HMDB) and the Kyoto
Encyclopaedia of Genes and Genomes (KEGG), yielding a total of
31 metabolites that were differentially regulated among WT, Tg
and GV-971-treated Tg mice, which could be matched to all three
databases (Supplementary information, Fig. S5g). Pathway enrich-
ment analysis of these metabolites using MBROLE or
MetaboAnalyst
47,48
further revealed signicant changes in amino
acid-related metabolic pathways and enzymes, especially
phenylalanine-related pathways (Fig. 5a).
We therefore chose to focus on amino acids for further study.
Plasma concentrations of a total of 36 amino acids were screened
in both WT and Tg mice (Supplementary information, Table S6).
Classication of these amino acids using a random forest
algorithm
49
ranked phenylalanine as the top hit that was different
between WT and Tg, followed by isoleucine, serotonin, histidine
and acetylornithine (Fig. 5b; Supplementary information, Fig. S5h),
suggested their strong correlation with the disease progression.
We then examined the concentration of the selected amino acids
in the faecal and blood samples in GV-971-treated or untreated Tg
mice, and compared it with that of WT mice. We found that the
concentrations of phenylalanine and isoleucine were signicantly
higher in the feces of Tg mice than those of WT mice, and GV-971
treatment signicantly reduced their concentrations to a level
comparable to that of WT mice (Fig. 5c). A similar change in the
concentration of phenylalanine and isoleucine was detected in
blood (Fig. 5d). To test whether the elevation of these amino acids
resulted from gut microbiota change, we examined the concen-
tration of amino acids in FMT study. Feces from 2-month-old WT
mice could signicantly reduce the concentration of phenylala-
nine and isoleucine in Tg mice (Supplementary information,
Fig. S5i). Likewise, in co-housed WT mice across various months of
age, phenylalanine and isoleucine concentrations in blood were
also elevated, comparable to that of Tg mice (Supplementary
information, Fig. S5j).
Amino acids could be taken by immune cells through
specic transporters, driving immune cell differentiation and
Fig. 3 The effects of GV-971 on behaviour changes in APP/PS1 mice models. aStructure of GV-971. GV-971 is a mixture of acidic linear
oligosaccharides with degrees of polymerization ranging from dimers to decamers with an average molecular weight of approximately 1 kDa.
bThe escape latency time results of the Morris Water Maze (MWM) test as a measurement of spatial learning and memory in APP/PS1 mice.
Nine-month-old APP/PS1 mice were treated with 50 mpk and 100 mpk of GV-971 for 3 months until 13-month old. Then, the MWM test for
spatial learning and memory abilities were conducted for 6 additional days. During the test, GV-971 was continuously administrated. The
escape latency time starting (seconds) was measured as one of the nal readouts of the test (see Materials and methods). Higher escape
latency time shows that these mice will spend more time to reach the target, which indicates a more severely impaired spatial learning and
memory ability (n=1114). The data are presented as mean ± standard error of the mean (mean ± SEM). Black asterisk indicates the
comparison between WT and APP/PS1 group. Blue asterisk indicates the comparison between GV-971(100 mpk) treatment and APP/PS1
group. *P< 0.05, ***P<0.001 by two-way ANOVA. cThe number of platform-site crossovers in MWM test as a measurement of spatial learning
and memory in APP/PS1 mice. Nine-month-old APP/PS1 mice were treated with 50 mpk and 100 mpk of GV-971 for 3 months until 13-month
old. Then, the MWM test for spatial learning and memory abilities were conducted for 6 additional days. During the test, GV-971 was
continuously administrated. The number of platform-site crossovers was measured as the other readout of the test (see Materials and
methods). Larger numbers of platform-site crossovers indicate less severely impaired spatial learning and memory ability (n=1117). *P<
0.05, ***P<0.001 by one-way ANOVA (F (3, 55) =6.542). dThe accuracy of spatial working memory as tested using the Y maze in APP/PS1
mice. Nine-month-old APP/PS1 mice were treated with 50 mpk and 100 mpk of GV-971 for 3 months until 12-month old. Then the Y maze test
was conducted. During the test, GV-971 was continuously administrated. The accuracy of the Y maze was the ratio between the
correct alternation and the total alternation (see Materials and methods). Higher accuracy indicates less severely inpaired working memory
abilities. (n=1720). The data are presented as mean ± standard error of the mean (mean ± SEM). **P< 0.01, ***P< 0.001 by one-way ANOVA
(F (3, 71) =12.39)
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Cell Research (2019) 0:1 – 17
proliferation.
50
We examined the expression levels of Slc7a5, the
transporter of phenylalanine and isoleucine, in Th1 cells and found
that Slc7a5 was expressed in Th1 cells (data not shown).
Furthermore, incubation of Th1 cells with
13
C-labelled phenylala-
nine revealed the uptake of phenylalanine by Th1 cells, which
could be blocked by a pharmacological inhibitor of SLC7A5
(Supplementary information, Fig. S5k), suggesting that Th1 cells
are prone to phenylalanine and isoleucine uptake.
To assess the direct effects of phenylalanine and isoleucine on T
cell differentiation, naïve CD4
+
T cells were exposed to either
phenylalanine or isoleucine with or without GV-971 for 4 days and
Th1 cells were assessed by Flow cytometry (FACS). We observed
the signicantly enhanced Th1 cell differentiation exposed to
either phenylalanine or isoleucine, which was inhibited by GV-971
treatment (Fig. 5e). Next, we evaluated the direct inuence of
phenylalanine and isoleucine on Th1 cell proliferation. We found
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Cell Research (2019) 0:1 – 17
that the proliferation of Th1 cells was signicantly promoted by
phenylalanine and isoleucine, and GV-971 treatment inhibited
phenylalanine-induced Th1 cell proliferation, but barely changed
isoleucines effect (Fig. 5f, blue column). Furthermore, we treated
WT mice with intraperitoneal injection of phenylalanine and
isoleucine and observed that Th1 cell frequency in the blood was
signicantly increased (Fig. 5g).
Finally, we explored whether the above ndings could be
recapitulated in MCI due to AD (see Materials and methodsfor
denition of MCI used in this study) patients. Indeed, phenylala-
nine and isoleucine concentrations as well as Th1 cell frequency in
the blood of MCI due to AD subjects were signicantly higher than
those in the age-matched healthy counterparts (Fig. 5h, i).
Moreover, the increased levels of both phenylalanine and
isoleucine in the blood were also conrmed in another small
MCI due to AD cohort (Fig. 5j), indicating that the abnormal
accumulation of phenylalanine and isoleucine and elevation of
Th1 frequency in the blood might function as a signature to
differentiate the MCI due to AD patients from the healthy subjects.
DISCUSSION
In this study, we revealed that dysbiosis of the gut microbiota is
required for the inltration of various peripheral immune cells,
including CD4
+
and CD8
+
T cells, B cells, natural killer (NK) cells,
neutrophil, dendritic cells (DCs) and monocytes, to the brain.
Among them, Th1 cells were particularly noted for the close
association with the M1 microglia activation during AD progres-
sion. Given the well-recognized functional crosstalk between Th1
and M1 microglia in the brain,
51
we propose that gut dysbiosis
promotes Th1 cell inltration to allow their local crosstalk with the
M1 microglia and in turn triggers the microglia differentiation
towards a pro-inammatory state.
This mechanistic insight is strengthened by a series of ndings
obtained in this study. Firstly, a dynamic shift of gut microbiota
composition during AD progression is signicantly correlated with
the elevation of Th1 cell inltration. Secondly, ablation of the gut
microbiota by antibiotic treatment blocked Th1 cells inltration
and M1 microglia activation in AD mice. Thirdly, both long-term
exposure to faecal bacteria (co-housing experiment) and FMT of
faecal bacteria from AD mice markedly enhanced Th1 cell
inltration and M1 microglia activation in WT mice, while reverse
FMT of WT mice feces into Tg mice reduced Th1 cells of the
recipient Tg mice. Our ndings collectively highlighted the gut
microbiota as a driving factor in promoting Th1/M1 microglia-
predominated neuroinammation in AD progression. Interest-
ingly, gut-derived inammatory immune cells such as Treg
and γδT were also reported to play roles in ischemic brain injury
52
and dietary salt-induced cognitive impairment,
53
which further
supports the functional link between gut microbiota and
neuroinammation and brain functions.
In our study, we did not observe any sex-dependent changes in
terms of gut microbiota composition or brain M1/Th1 cell
abundance, which seemed different from recent ndings showing
sex-dependent changes of microglia and Aβdeposition under
antibiotic treatment.
54
We speculate that this discrepancy might
result from the different experimental settings, including mouse
models (APP/PS1-21 vs. 5XFAD), antibiotic treatment periods at
different ages of mice and readout parameters.
The mechanistic relationship regarding gut microbiota dysbiosis
and neuroinammation in AD remains unclear, though advance-
ments have recently been made in other neurological disorders
including Parkinsons disease (PD). Previous studies suggest the
direct or indirect involvement of bacterial metabolites, in
particular, short-chain fatty acid (SCFA) and bile acids
32,55
in some
neurological disorders. In this study, we detected many metabo-
lites signicantly altered in AD mice compared to WT mice.
Among them, the most signicant change occurred in amino
acids, particularly those in phenylalanine-related pathways. We
conrmed that the abundance of phenylalanine and isoleucine
was increased in the feces and blood of AD mice compared to that
of WT mice. Functional assessment both in vitro and in vivo
revealed the role of phenylalanine and isoleucine in promoting
both differentiation and proliferation of peripheral inammatory
Th1 cells. These ndings highlight the role of the abnormal
production of phenylalanine and isoleucine by gut microbiota in
provoking Th1 cell-dominated neuroinammation. Consistent
with this notion, we detected higher concentration of phenyla-
lanine/isoleucine and Th1 cell frequency in the blood of MCI due
to AD patients compared with that of the healthy control cohort.
All these ndings allowed us to propose a conceptual
advancement in understanding the mechanism of AD
Fig. 4 GV-971 alleviates neuroinammation by reconditioning the gut microbiota. aPrincipal coordinate analysis (PCoA) of the gut
microbiome composition on the operational taxonomic unit (OTU) level based on the Bray-Curtis distance for 5XFAD (Tg) mice and GV-971-
treated Tg mice at 7-month old (n=7). The shapes and colours of the points indicate samples from each individual. Coloured ellipses indicate
0.95 condence interval (CI) ranges within each tested group. PC principal component. bHeatmap of signicant gut microbiota changes
represented at the genus level between 5XFAD (Tg) mice and GV-971-treated Tg mice at 7-month old (n=7). Colours on the heatmap indicate
the relative abundance of gut microbiota; red indicates bacteria that are upregulated, and blue indicates bacteria that are downregulated. Gut
microbiota with signicant changes were chosen using a Wilcoxon rank-sum two-tailed test with P-value that is less than 0.05 between Tg and
GV-971-treated groups. f family, o order. cChanges in correlational links between the gut microbiome at the genus level (designated with
numbers near the purple circles) and brain lymphocytes (other coloured circles) before (left) and after (right) oral gavage of GV-971 in
7-month-old 5XFAD (Tg) mice (n=57). Lines represent either signicant (P-value < 0.05) positive or negative correlation (Pearson coefcient).
The size of each circle of immune cells are positively related to the links connected to this circle. The right side lists the name of each gut
microbiome, also see Materials and methods. f family, o order, DC dendritic cells, NK natural killer cells, Neu neutrophils, B B cells, Mo/Mϕ
monocytes and macrophages. dEffect of GV-971 treatment on the frequency of brain Th1 cells in 5XFAD (Tg) mice at 7 months old (n=57).
Th1 cell counts (CD45
high
CD4
+
CXCR3
+
) are presented relative to CD45
high
CD4
+
T cell counts detected by ow cytometry. The data are
presented as mean ± standard error of the mean (mean ± SEM). *P<0.05, ***P< 0.001, by Students t-test. eEffect of GV-971 treatment on the
positive signal density of IBA1 immunouorescent staining detected in hippocampal slices from 5XFAD (Tg) mice at 7-month old, reecting
activation of microglial cells (n=46). The data are presented as mean ± standard error of the mean (mean ± SEM). **P<0.01, ***P<0.001, by
one-way ANOVA (F (2, 15) =21.94). fEffect of GV-971 treatment on levels of cytokine proteins in the brain homogenates of 5XFAD (Tg) mice at
7-month old as detected by a cytokine antibody array (n=56). Colours on the heatmap indicate relative cytokine levels; red indicates
cytokines that are upregulated, and blue indicates cytokines that are downregulated. g, h Effect of GV-971 on Aβ-positive area (g) and tau-
positive area (h) in the hippocampus of 5XFAD (Tg) mice at 7-month old, evaluated in brain slices (n=47). The data are presented as mean ±
standard error of the mean (mean ± SEM). For Aβanalysis: *P<0.05, ***P<0.001 (F (2, 14) =22.78). For tau analysis: *P<0.05, ***P<0.001 (F (2,
15) =13.06) by one-way ANOVA. iEffects of GV-971 on the time out of 10
4
sec (s) taken to achieve 80% success (see Materials and methods)
in a test to evaluate the discrimination learning abilities of 5XFAD (Tg) mice at 7-month old (n=1013). Time means the time to reach the 80%
performance level (seconds); the longer it takes, the severer the cognitive impairment is (see Materials and methods). *P<0.05, ***P<0.001
by One-way ANOVA (F(2,31) =9.751). The concentration of GV-971 was 100 mpk for all of the above results
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Cell Research (2019) 0:1 – 17
pathogenesis. AD is not only an Aβ-driven brain disease. Its
development also requires the systematic interaction between
gut, brain and the intermediate inammatory factors. In the
context of Aβdeposition, the altered gut microbiota composition
during AD progression causes an abnormal elevation of amino
acids, phenylalanine and isoleucine in particular. These amino
acids promote the inltration of peripheral Th1 cells into the brain
via blood circulation. The inltrating peripheral Th1 cells may
locally crosstalk with M1 microglial cells in the brain, resulting in
pathological neuroinammation and cognitive impairment (Fig. 6).
These mechanistic insights into AD pathogenesis may provide a
novel therapeutic solution by reconstituting the gut microbiota to
favour anti-neuroinammation responses.
Emerging data have shown that poly- or oligosaccharides
have advantages to modulate gut microbiota.
56
The oligoman-
nate sodium GV-971 is a carbohydrate-based anti-AD drug that
has been demonstrated to reverse cognitive impairment in mild-
to-moderate AD patients in a recently completed phase 3
clinical trial in China. GV-971 is well tolerated with a safety
prole comparable to the placebo control. In this study, we
found that GV-971 effectively reconditioned gut microbiota,
decreased the concentration of phenylalanine and isoleucine in
the feces and blood, and reduced Th1-related neuroinamma-
tion in the brain. Of note, GV-971-treated Tg feces could largely
mimic the therapeutic effects of GV-971 treatment per se, while
antibiotic treatment abrogated the therapeutic effects of GV-
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Fig. 5 GV-971 inhibits neuroinammation by harnessing amino acid metabolism. aPathway enrichment analysis of faecal metabolites in 7-
month-old 5XFAD (Tg) mice with or without GV-971 treatment (100 mpk) using MBROLE (n=68). A partial ist of the enrichment results is
presented with KEGG modules and KEGG enzyme interactions which have been screened using a criterion of FDR-adjusted P-value < 0.05. b
Lists of the most important blood amino acids of the random forest model ranked from most to least important between WT (2 m-9 m) and Tg
(2 m-9 m) group from a ROC curve analysis. Red indicated high concentration, green indicated low concentration. (n=30 for WT, n=26 for
Tg). cChanges in histidine, phenylalanine and isoleucine levels in the feces of WT, 5XFAD mice (Tg), and GV-971-treated Tg mice (100 mpk) (n
=611) at 7-month old. Colours in the heatmap indicate relative metabolite levels; red indicates metabolites that are upregulated, and blue
indicates metabolites that are downregulated. dChanges in histidine, phenylalanine and isoleucine levels in the blood of WT, 5XFAD mice
(Tg), and GV-971-treated Tg mice (100 mpk) (n=67) at 7-month old. Colours in the heatmap indicate relative metabolite levels. Red indicates
metabolites that are upregulated, and blue indicates metabolites that are downregulated. eThe effects of GV-971 on the differentiation of
naïve CD4
+
T cells (Th0 cells) to Th1 cells induced by phenylalanine and isoleucine in vitro. Naïve CD4
+
T cells were cultured for 5 days with/
without GV-971 in the presence of phenylalanine (1 mM) or isoleucine (1 mM). The frequency of Th1 (CD4
+
IFN-γ
+
) cells was tested by ow
cytometry (see Materials and methods). GV-971 was used at a nal concentration of 100 µg/mL. The data are presented as mean ± standard
error of the mean (mean ± SEM); n=3 replicates per group, one of three replicated results was represented. Left, *P<0.05, **P<0.01 by one-
way ANOVA (F (2, 6) =15.64). Right, *P<0.05, **P<0.01 by one-way ANOVA (F (2, 6) =10.35). fThe effects of GV-971 on the proliferation of
Th1 cells induced by phenylalanine and isoleucine. Th1 cells were stained with CellTrace and cultured for 4 days with/without GV-971 in the
presence of phenylalanine (1 mM) and isoleucine (1 mM). The density of CellTrace uorescence in Th1 (CD4
+
IFN-γ
+
) cells was tested by ow
cytometry (see Materials and methods). GV-971 was used at a nal concentration of 100 µg/mL. The data are presented as mean ± standard
error of the mean (mean ± SEM), n=3 replicates per group, one of three replicated results was represented. *P<0.05, ***P<0.001 by one-way
ANOVA (F (4, 9) =28.34). Phe, phenylalanine; Ile, isoleucine. gFrequency of blood Th1 cell changes in C57 mice after 4-day intraperitoneal (i.p.)
injection of phenylalanine and isoleucine (n=8). ***P<0.001 by one-way ANOVA (F (2, 21) =101.8). hRandom forest classication of amino
acid changes in healthy controls (HC) and mild cognitive impairment (MCI) due to AD patients. The amino acids are ranked by mean decrease
in classication accuracy (rst cohort, n=9 for MCI due to AD, n=18 for HC). Red indicated high concentration, green indicated low
concentration. iFrequency of Th1 cells in the blood of healthy controls (HC) and mild cognitive impairment (MCI) due to AD patients (rst
cohort, n=8 for MCI due to AD, n=9 for HC). *P<0.05 by Students t-test. jLevels of phenylalanine and isoleucine in the blood of healthy
controls (HC) and mild cognitive impairment (MCI) due to AD patients (second cohort, n=22 for both groups). *P< 0.05 by Students t-test
Fig. 6 Schematic diagram of gut-brain axis in AD progression and the intervention strategy. Along with Aβdeposition and tau
phosphorylation, the alteration of the gut microbiota during AD progression causes metabolic disorder. The abnormal metabolites production
provoke peripheral inammation, increases the brain inltration of immune cells which crosstalk with M1 microglial cells in the brain, resulting
in pathological neuroinammation and cognitive impairment (left panel). Oral administration of GV-971 reconditions the gut microbiota,
normalizes disordered metabolites, reduces the peripheral immune cell inltration to the brain, resolves neuroinammation, and reduces Aβ
deposition and tau phosphorylation, leading to ultimate improvement of cognitive functions (right panel)
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Cell Research (2019) 0:1 – 17
971. These ndings provided important evidence showing that
the therapeutic effect of GV-971 is primarily via the reconstitu-
tion of gut microbiome. As such, GV-971 may provide us an
appealing approach of microbiota-centric anti-AD strategies
worthy of future investigations.
Our ndings may have translational implications for AD
diagnosis and therapy. The combination of specicbacteria
(e.g., Th1/M1-associated bacteria), amino acids (e.g., phenylala-
nine and isoleucine) and brain inltrating immune cell
composition (e.g., Th1 dominant) could be used as an early
diagnostic biomarker for MCI due to AD patients, which merits
further validation in large cohorts of AD patients. More
importantly, the identied microbiota-centric anti-AD effect of
GV-971 will open a new therapeutic avenue for AD treatment
through remodelling gut microbiota, and guide the future
development of effective therapies by exploring the enormous
chemical space of carbohydrates.
Meanwhile, our study has several limitations. Though we have
revealed the potential role of gut microbiota in the pathogenesis
of AD, future studies to identify the key bacteria strains that may
account for metabolites and neuroinammation changes will be
critical. Beyond the two key amino acids that have been fully
studied in this study, further exploration will be required to
understand other mechanistic links connecting gut microbiota
and neuroinammation. Future studies to address these key issues
may help gain a better understanding of the pathogenesis of AD
and explore the broader therapeutic potential of GV-971 in clinical
treatment.
MATERIALS AND METHODS
GV-971
GV-971 is a mixture of oligosaccharides with the degree of
polymerization (dp) from 2 to 10. The non-clinical study showed
that GV-971 penetrates the blood-brain-barrier (BBB) in its
original form. Type 1 glucose transporter (GLUT1) was identied
as one of the transporters accounting for its penetration
mechanism of BBB. GV-971 could directly bind to multiple
subregions of Aβto inhibit Aβbril formation and destabilize
the preformed brils into non-toxic monomers. Through
targeting Aβ, GV-971 promoted microglia-mediated Aβphago-
cytosis in vitro, and reversed cognition impairment in multiple
AD models. All the preclinical pharmacology, PD/PK and
toxicology studies were completed.
GV-971 exhibited a safe and well-tolerated prole in both phase
1 and phase 2 trials. In the recently completed phase 3 trial
(NCT02293915), GV-971 was demonstrated to meet the primary
endpoint, with statistic signicance (p < 0.001). No serious adverse
events were observed, with similar incidence rate between GV-971
and placebo group (unpublished data). We are currently applying
for NDA ling for GV-971 in China.
Animals
5XFAD (Tg) mice and co-housed WT mice (the corresponding WT
mice generated by mating Tg mice and C57 mice) were bred in
the same cage after birth. The WT mice (C57) were bred in
separate cages to avoid microbiota cross transfer. All the mice
were maintained in a room at 23 °C under a 12-hour (h) light-dark
cycle. Mice were randomly allocated to different groups before
treatment. For time course analysis of Tg mice, male and female
Tg mice were sacriced at 2-, 3-, 5-, 7- and 9-month old. Mouse
brains were collected and stained for immune cell analysis and
cytokine analysis. Before the mice were sacriced, feces were
collected for gut microbiota analysis. For GV-971 treatment, at 7-
month old, Tg mice were treated with GV-971 at 100 mg/kg dose
by oral gavage once daily for one month based on 450 mg twice
per day in the Phase 3 trial. A behavioral test was performed to
monitor cognitive activity after the last treatment. Then, the
mouse brains and feces were used for different analyses. For
behavior test, Tg mice and WT mice at different months as well as
mice after treatment were tested by discrimination learning, as
reported previously.
57
For phenylalanine and isoleucine intraper-
itoneal injection treatment, 4-month-old C57 mice were treated
with phenylalanine or isoleucine at 50 mg/kg for 4 days.
For Morris water maze (MWM) and Y maze tests, APP/PS1 mice
were dosed orally with GV-971 at 50 and 100 mg/kg/day
beginning at 9 months. Two tests were carried out at 13 months.
For APP/PS1 mice time point analysis, feces were collected at
various months of age before we sacriced the mice for immune
cell analysis. The mice and age-matched wild-type mice were
sacriced at 3-, 9-, and 14-month old for immune cell analysis.
Mouse brains and blood were collected for different analyses. For
APP/PS1 mice treated with GV-971 and antibiotics, 6-month-old
mice were treated with GV-971 at 50 mg/kg by oral gavage once
daily with or without antibiotic treatment for 2 months. The
animal experiments were approved by the ethical committee of
Shanghai SIPPR-Bk Lab Animal Co., Ltd (Number: 2016002) and by
the Institutional Animal Care and Use Committee at Shanghai
Institute of Materia Medica, China.
Behavioral tests
The MWM test is used to measure spatial learning and memory
according to a protocol published previously.
58
Briey, the mice
underwent 6-day acquisition experiments, and each mouse
performed 3 trials each day. The animals were released into the
water at the desired start position, and the latency to nd the
platform was timed. On the 7
th
day, the platform was removed,
and the mice were allowed to swim for 60 s. The trace and the
number of platform-site crossovers were recorded using a video
camera.
Working memory was assessed by the Y maze according to the
literature with some modications.
58
The Y maze was composed
of three identical arms (A, B, C) with different cues. Mice were
placed in the start arm (A) and the sequence of explored arms was
recorded (such as ABCBA). The total number of arm entries and
alternation behavior were recorded using a video camera. The
accuracy of the Y maze was the ratio between the correct
alternation and the total alternation.
Discrimination learning. 5XFAD mice and WT mice at different
months as well as mice after treatment were tested by Cognition
Wall discrimination spatial learning task, as reported previously.
57
Mice were habituated to individual cages one week before the
test. Approximately, 6 h before the test (10:00 am), the mice were
housed in the automated PhenoTyper (HomeCage) to habituate to
the cage. During the habituation phase, the mice had free access
to water but limited access to food. The task began at 4:00 pm and
continued during the night for 16 h. During the task, mice need to
learn to enter the Cognition Wall through the left entrance (three
entrances were offered) to obtain food (one food pellet was
rewarded for every fth entry through the left entrance). The total
time needed to reach a criterion of 80% correct, computed as a
moving window with window size equal to 30 (i.e., 24 correct
entries out of the 30 last entries), was used as a measure during
the task. All mouse movements were recorded by a computerized
tracking system (Noldus, Ethovision). More time to reach the 80%
correct criterion reects less discriminative ability.
Faecal sample DNA extraction, PCR amplication and sequencing
All faecal samples were frozen at 80 °C before DNA extraction
and analysis. The following steps were conducted by Majorbio Bio-
Pharm Technology Co., Ltd. (Shanghai, China). Microbial DNA was
extracted from faecal samples using the E.Z.N.A.®Soil DNA Kit
(Omega Bio-Tek, Norcross, GA, U.S.) according to the manufac-
turers protocols. The nal DNA concentration and purication
Article
11
Cell Research (2019) 0:1 – 17
were determined by a NanoDrop 2000 UV-vis spectrophotometer
(Thermo Scientic, Wilmington, USA), and DNA quality was
checked by 1% agarose gel electrophoresis. The V3-V4 hypervari-
able regions of the bacterial 16S rRNA gene were amplied with
primers 338 F (5-ACTCCTACGGGAGGCAGCAG-3) and 806 R (5-
GGACTACHVGGGTWTCTAAT-3) by a thermocycler PCR system
(GeneAmp 9700, ABI, USA). PCR reactions were conducted using
the following program: 3 min (min) of denaturation at 95 °C, 27
cycles of 30 sec (s) at 95 °C, 30 s for annealing at 55 °C, and 45 s for
elongation at 72 °C, and a nal extension at 72 °C for 10 min. PCR
reactions were performed in triplicate in a 20 μL mixture contain-
ing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mmol/L dNTPs, 0.8 μLof
each primer (5 μmol/L), 0.4 μL of FastPfu Polymerase and 10 ng of
template DNA. The resulting PCR products were extracted from a
2% agarose gel and further puried using the AxyPrep DNA Gel
Extraction Kit (Axygen Biosciences, Union City, CA, USA) and
quantied using QuantiFluor-ST (Promega, USA) according to the
manufacturers protocol. Puried amplicons were pooled in
equimolar and paired-end sequenced (2 × 300) on an Illumina
MiSeq platform (Illumina, San Diego, USA).
Processing of sequencing data
The following steps were conducted by Majorbio Bio-Pharm
Technology Co., Ltd. (Shanghai, China). Raw fastq les were
demultiplexed, quality ltered by Trimmomatic and merged by
FLASH based on the following criteria: (i) The reads were truncated
at any site that received an average quality score < 20 over a 50 bp
sliding window. (ii) The primers were exactly matched, allowing a
2-nucleotide mismatch, and reads containing ambiguous bases
were removed. (iii) Sequences with overlaps of longer than 10 bp
were merged according to their overlap sequence. Operational
taxonomic units (OTUs) were clustered with a 97% similarity cutoff
using UPARSE (version7.1 http://drive5.com/uparse/), and chimeric
sequences were identied and removed using UCHIME. The
taxonomy of each 16S rRNA gene sequence was analyzed by the
RDP Classier algorithm (http://rdp.cme.msu.edu/) against the
Silva 16S rRNA database (silva 132/16s bacteria) using a
condence threshold of 70%.
Metabolites sample preparation
Samples stored at 80 °C were taken out and thawed at room
temperature. The following steps were conducted by Majorbio Bio-
Pharm Technology Co., Ltd (Shanghai, China). In the experiment, 50
mg samples were used, and 400 μL methanol-water (4:1, v/v) were
also added to homogenize the sample using a homogenizer for 10s.
The solution was ultrasonically extracted on ice for 10 min and
stored at 20 °C for 30 min, then centrifuged for 15 min at 13000
rpm at 4 °C. For LC-MS analysis, 200 μLsupernatantwasused.QC
sample was prepared by mixing aliquots of all samples to be a
pooled sample and then analyzed using the same method with the
analytic samples. The QCs were injected at regular intervals (every
10 samples) throughout the analytical run to provide a set of data
from which repeatability can be assessed.
LC/MS analysis parameters
The following steps were conducted by Majorbio Bio-Pharm
Technology Co., Ltd. (Shanghai, China). LC-MS was performed on
AB Sciex TripleTOF 5600TM mass spectrometer system (AB SCIEX,
USA). LC Conditions: Column: Acquity BEH C18 column (100 mm ×
2.1 mm i.d., 1.7 µm; Waters, Milford, USA). Solvent: The column was
maintained at 40 °C and separation was achieved using the
following gradient: 5% B30% B over 03 min, 30% B95% B over
39min,95%B95% B over 913.0 min; 95% B5% B over 13.013.1
min, and 13.116 min holding at 5 % B at a ow rate of 0.40 mL/min,
where B is acetonitrile: isopropanol 1:1 (0.1% (v/v) formic acid) and A
is aqueous formic acid (0.1% (v/v) formic acid). Injection Volume was
20 μL. The mass spectrometric data was collected using an AB Sciex
TripleTOF 5600TM mass spectrometer system equipped with an
electrospray ionization (ESI) source operating in either positive or
negative ion mode with a capillary voltage 1.0 kV, sample cone, 40 V,
collision energy 6 eV. The source temperature was set at 120 °C, with
a desolvation gas ow of 45 L/h. Centroid data was collected from
50 to 1000 m/z with a 30000 resolution.
In vitro differentiation of naïve CD4
+
T to Th1 and Th2 induced by
the supernatant of mice feces
We used a previously well-established protocol for the in vitro
culture of fecal samples and collected the supernatant.
4446
Briey,
gut feces of 7-month-old mice were collected, put into the culture
medium (with or without GV-971, nal concentration of 100 µg/
mL) and 96 deep-well plates (2 mL total volume) at 37 °C on the
shaking bed (500 rpm) inside the anaerobic incubator for a total
culture period of 24 h before we collected the gut-microbiota-
derived metabolites-containing supernatant. Naïve CD4
+
T cells
were separated from the splenocytes of 8-month-old C57BL/6
female mice using the naïve CD4
+
T cell Isolation Kit (Stem Cell,
Cat No. 19765). A total of 0.5 × 10
6
cells/well in 0.5 mL of RPMI-
1640 medium were plated in 48-well anti-CD3 and anti-CD28 pre-
coated plates, and the culture medium was supplemented with
cytokines and blocking antibodies. Th0: 20 ng/mL mIL-2; Th1: 20
ng/mL mIL-2, 10 μL supernatant, 5000 ng/mL 11B11; Th2: 20 ng/
mL mIL-2, 10 μL supernatant, 5000 ng/mL XMG1.2. After incuba-
tion at 37 °C in 5% CO
2
for 5 days, cells were stimulated with Cell
Activation Cocktail (Thermo sher, #00-4975-03) in 5% CO
2
at 37 °
C for 4 h, washed twice with PBS, labeled with zombie yellow
(Biolegend, # 423104) to exclude dead cells. Non-specic binding
of immunoglobulin to the Fc receptors was blocked with anti-
CD16/32 (Biolegend, #101320). Intracellular staining was per-
formed with anti-CD4 (Biolegend, #100406), anti-IFNγ(Biolegend,
#505826) and anti-IL4 (Biolegend, #504104) according to the
manufacturesprotocal. Cells were acquired on a BD Aria III
cytometer, and data were analyzed using FlowJo software.
Immunohistochemistry
For 3,30-diaminobenzidine (DAB)-developed staining, sections
were immunostained using a Leica BOND-RX Autostainer (Leica
Microsystems) and Coverslipper CV5030 (Leica Microsystems)
according to the manufacturers IHC protocol. Briey, sections
were submitted to heat-induced epitope retrieval with Epitope
Retrieval solution 2 (ER2, AR9640, Leica Biosystems) for 20 min.
Endogenous peroxidase activity was blocked with 3%-4% (v/v)
hydrogen peroxide (part of DS9800, Leica Biosystems) for 10 min.
Then, sections were incubated with blocking buffer (10% NGS in
PBS with 0.3% Triton x-100) for 60 min. Finally, staining was
performed using the Bond Polymer Rene Detection System
(DS9800, Leica Biosystems) according to the manufacturers
protocol. The primary antibody incubation time was 30 min.
Sections were stained for activated microglia using rabbit anti-
IBA1 antibody (1:1,000, cat# 019-19741, Wako), amyloid deposi-
tions using mouse anti-Aβ42 antibody (1:1,000; cat# 803003,
Biolegend) and Tau phosphorylation using mouse anti-PHF-Tau &
tangles -Thr231 antibody (1:300, cat# MN1040, Thermo Fisher).
Stained slices were automatically scanned by a high-throughput
bright eld scanner (NanoZoomer 2.0HT, Hamamatsu), and images
were obtained by NDP.scan 3.2 software (Hamamatsu). For
uorescent staining, slides were blocked by blocking buffer
(10% NGS in PBS with 0.3% Triton x-100) at RT for 1 h, and then
incubated in the primary antibody solution (Iba-1 1:1,000, Aβ42
1:1,000, Tau 1:300) overnight at 4 °C. After washing, slides were
incubated with uorescent anti-rabbit or anti-mouse secondary
antibody (1:1000, Invitrogen) for 60 min at RT and further washed
3 times in PBS. Finally, slides were counterstained with DAPI
(1:10000 in PBS, Sigma) for 5 min at RT, washed, sealed and stored
at 4 °C for image acquisition. Representative uorescence images
were acquired by upright uorescence microscope Zmager-m2
(Zeiss, Germany) under 10× objective using Zen software (Zeiss).
Article
12
Cell Research (2019) 0:1 – 17
Amino acid detection
A set of amino acid standard mixture solutions was prepared at a
concentration range of 1002000 μmol/L. A portion of 10 μLof
each standard mixture solution or plasma sample was pipetted
into the bottom of a tube, and then 70 μL of sodium borate buffer
(200 mmol/L at pH 8.8) was added. After 20 μL of 6-aminoquinolyl-
N-hydroxysuccinimidyl carbamate (AQC) (4 mg/mL in acetonitrile)
was added, the tube was closed and heated for 10 min at 55 °C to
form AQCamino acid. The solution was then cooled down to
room temperature and 2 μL portion of each solution was injected
into the UPLC-ESI-MS system for amino acid analysis without
further purication.
Human subjects
The blood from MCI due to AD patients and healthy control was
collected from a pilot study. MCI due to AD is dened in the NIA-
AA 2011 clinical and research diagnostic criteria for MCI due to AD.
The patients with MCI due to AD in this study must meet the
following criteria. First, concern regarding a change in cognition.
Second, impairment in one or more cognitive domains. Third,
preservation of independence in functional abilities. Forth, not
demented (NIA-AA 2011 clinical diagnosis criteria for MCI due to
AD). All participants underwent a screening process that included
a review of their medical history, physical and neurological
examinations, laboratory tests, and MRI scans. The clinical
assessment of mild cognitive impairment or dementia included
neuropsychological tests, as well as behavioral and psychiatric
interviews conducted by attending psychiatrists. AD patients
recorded scores of < 4 on the Hachinski Ischemia Scale and
showed no history of signicant systemic or psychiatric condi-
tions, or traumatic brain injuries that could compromise brain
function. The Clinical Dementia Rating (CDR) and Montreal
Cognitive Assessment (MoCA) were assessed for all of the
participants. Based on the assessment, we retained MCI due to
AD subjects and others were excluded such as those who had
impairment in a single non-memory domain (single, non-memory
domain MCI subtype) and those who had impairment in two or
more domains (multiple domains, slightly impaired MCI subtype).
Normal control subjects had no history of cognitive decline,
neurological disorders, or uncontrolled systemic medical disor-
ders. All subjects included in this study had no more than two
lacuna ischemia (of diameter < 1 cm) as revealed by MRI uid-
attenuated inversion recovery (FLAIR) sequence.
The sample size for the rst cohort (Fig. 5h, i) is 9 MCI due to AD
patients and 18 healthy subjects. The sample size for the second
cohort (Fig. 5j) is 22 MCI due to AD patients and 22 healthy
subjects. A diagnosis of MCI was based on the following criteria,
which were adapted from the MCI diagnostic criteria of Petersen:
(1) memory complaints, preferably corroborated by a spouse or
relative; (2) objective memory impairment; (3) normal general
cognitive function; (4) intact activities of daily living; and (5)
absence of dementia. The Ethics Committee of the Shanghai
Mental Health Centre Institutional Review Board approved the
study (Number: 2016-22R1). Informed consents were obtained
from the subjects and the guardian of the subjects. Information
about gender and age etc. are provided below.
First cohort
Type Sample ID Age Gender
Healthy HC-01 75 F
Healthy HC-02 71 F
Healthy HC-04 69 F
Healthy HC-05 73 F
Healthy HC-08 63 M
Healthy HC-09 62 M
Type Sample ID Age Gender
Healthy HC-10 68 M
Healthy HC-11 61 M
Healthy HC-12 68 M
Healthy HC-13 75 F
Healthy HC-14 70 F
Healthy HC-15 68 M
Healthy HC-16 62 M
Healthy HC-17 75 M
Healthy HC-18 65 F
Healthy HC-19 64 F
Healthy HC-20 64 F
Healthy HC-22 63 F
MCI due to AD MCI-02 61 F
MCI due to AD MCI-03 79 M
MCI due to AD MCI-04 71 F
MCI due to AD MCI-05 72 F
MCI due to AD MCI-06 80 F
MCI due to AD MCI-07 65 M
MCI due to AD MCI-09 61 M
MCI due to AD MCI-10 69 M
MCI due to AD MCI-14 77 M
Second cohort
Category Sample ID Age Gender
Healthy HC-0149 73 M
Healthy HC-0150 70 F
Healthy HC-0153 63 F
Healthy HC-0156 67 M
Healthy HC-0158 71 F
Healthy HC-0165 61 F
Healthy HC-0168 62 F
Healthy HC-0172 70 F
Healthy HC-0176 71 F
Healthy HC-0187 73 F
Healthy HC-0191 65 M
Healthy HC-0013 70 F
Healthy HC-0043 81 M
Healthy HC-0050 64 F
Healthy HC-0051 66 M
Healthy HC-0056 75 M
Healthy HC-0068 67 F
Healthy HC-0074 57 F
Healthy HC-0076 60 F
Healthy HC-0081 71 F
Healthy HC-0089 57 F
Healthy HC-0090 76 F
MCI due to AD MCI-0169 65 F
MCI due to AD MCI-0175 61 F
MCI due to AD MCI-0211 69 M
MCI due to AD MCI-0229 65 M
MCI due to AD MCI-0240 59 F
MCI due to AD MCI-0244 64 F
Article
13
Cell Research (2019) 0:1 – 17
Category Sample ID Age Gender
MCI due to AD MCI-0257 61 F
MCI due to AD MCI-0260 60 F
MCI due to AD MCI-0282 66 F
MCI due to AD MCI-0284 64 F
MCI due to AD MCI-0287 75 F
MCI due to AD MCI-0303 62 F
MCI due to AD MCI-0319 63 F
MCI due to AD MCI-0332 67 F
MCI due to AD MCI-0374 61 M
MCI due to AD MCI-0385 68 F
MCI due to AD MCI-0401 57 F
MCI due to AD MCI-0414 64 F
MCI due to AD MCI-0001 75 M
MCI due to AD MCI-0043 55 F
MCI due to AD MCI-0061 63 F
MCI due to AD MCI-0091 78 F
Th1 cell staining in human samples
Two milliliters of peripheral blood from healthy people or patients
were treated with red blood cells lysis buffer. A total of 1 × 10
6
cells were collected and incubated in 100 μL of PBS containing
Zombie Yellow Dye (Biolegend, #423104) for 15 min to label live
cells. After washing, samples were stained with APC/Cy7-
conjugated anti-human CD45 (Biolegend, #304014), Alexa Fluor
700-conjugated anti-human CD3 (Biolegend, #344822), FITC-
conjugated anti-human CD4 (Biolgend, #300506), Brilliant Violet
421-conjugated anti-human CXCR3 (Biolgend, #353716) and APC-
conjugated anti-human CCR6 (Biolgend, #353416) for 30 min.
Stained cells were washed twice with PBS. Cells were acquired
using FACS Fortessa X-20 ow cytometer (BD Biosciences, San
Jose, CA). Live CD45
+
CD3
+
CD4
+
CXCR3
+
CCR6
-
cells were gated as
Th1 subsets as previously described.
59
Data analysis were
performed with owjo software (version 10.5, LLC)
In vitro differentiation induced by phenylalanine and isoleucine
As previously described,
60
naïve CD4
+
T cells were enriched from
the splenocytes of 8-month-old C57BL/6 female mice using the
Naïve CD4
+
T cell Isolation Kit (Stem Cell, #19765). For cell
differentiation assay, a total of 1 × 10
5
cells/well in 0.2 mL of RPMI-
1640 medium were plated in anti-CD3/CD28-coated 96-well plates
(anti-CD3, 2 μg/mL; anti-CD28, 1 μg/mL), and the culture medium
was supplemented with cytokines or blocking antibodies. Th0:
mIL-2 (10 ng/mL); Th1: mIL-2 (10 ng/mL) and 11B11 (5000 ng/mL).
Phenylalanine (nal concentration, 1 mmol/L), isoleucine (nal
concentration, 1 mmol/L) and GV-971 (nal concentration, 100 µg/
mL) were added into the indicated wells, respectively. After
incubation at 37 °C in 5% CO
2
for 5 days, cells were stimulated
with Cell Activation Cocktail (Thermo sher, #00-4975-03) in 5%
CO
2
at 37 °C for 4 h, washed twice with PBS, and labeled with
zombie yellow (Biolegend, #423104) to exclude dead cells. Non-
specic binding of immunoglobulin to the Fc receptors was
blocked with anti-CD16/32 (Biolegend, #101320). Intracellular
staining was performed with anti-CD4 (Biolegend, #100406) and
anti-IFNγ(Biolegend, #505830) according to the manufactures
protocal. Cells were acquired on a BD Fortessa cytometer, and
data were analyzed using FlowJo software.
In vitro proliferation induced by phenylalanine and isoleucine
A total of 1 × 10
6
naïve CD4
+
T cells were dissolved with 3 mL of
Th1 differentiation Medium (10 ng/mL of mIL-2, 20 ng/mL of mIL-
12 and 5000 ng/mL of 11B11) and plated in anti-CD3/CD28-coated
60-mm culture dish (anti-CD3, 2 μg/mL; anti-CD28, 1 μg/mL). The
cells were incubated in 5% CO
2
at 37 °C for 5 days.
Next, ow cytometry was applied to gate live cells (Zombie
yellow dye, Biolegend, #423104) and CD4
+
CXCR3
+
Th1 cells were
sorted by anti-CD4 (Biolegend, #100406) and anti-CXCR3 (Biole-
gend, #126522) staining. The viability of sorted Th1 cells was
further conrmed by Trypan blue staining.
The sorted Th1 cells was stained with CellTrace (Thermo Fisher,
#C34557) and plated in anti-CD3/CD28-coated microplate and
cultured in RPMI-1640 medium in the presence of phenylalanine
(nal concentration, 1 mmol/L), isoleucine (nal concentration, 1
mmol/L) and GV-971 (nal concentration, 100 µg/mL). After
incubation in 5% CO
2
at 37 °C for 4 days, cells were stimulated
with Cell Activation Cocktail (Thermo sher, #00-4975-03) in 5%
CO
2
at 37 °C for 4 h, washed twice with PBS, and labeled with
zombie yellow (Biolegend, #423104) to exclude dead cells. Non-
specic binding of immunoglobulin to the Fc receptors was
blocked with anti-CD16/32 (Biolegend, #101320). Intracellular
staining was performed with anti-CD4 (Biolegend, #100422) and
anti-IFNγ(Biolegend, #505806) according to the manufactures
protocal. Cells were acquired on a BD Fortessa cytometer, and
data were analyzed using FlowJo software.
Uptake of phenylalanine
Naïve CD4
+
T cells were separated from the splenocytes of 8-
month-old C57BL/6 female mice using the Naïve CD4
+
T cell
Isolation Kit (Stem Cell, Cat No. 19765) and were induced to Th1
differentiation by 20 ng/mL IL-12. After 3 days, a total of 5 × 10
5
cells/well Th1 cells in 0.5 mL of RPMI-1640 medium were plated
into 48-well plates.
13
C-labelled phenylalanine and 5 mM amino
transporter inhibitor BCH (CAS:20448-79-7) were added into
indicated wells. After 0.5 h, cells were collected and washed twice
with ice-cold D-PBS. Then, 50 µL deionized water was added and
cells were lysed through freezing and thawing twice at 80 °C.
The cell lysate was centrifuged at 12000 × gfor 10 min and
13
C-
labelled phenylalanine in the supernatant was analyzed by Mass
spectrometry.
Antibiotic treatments
Mice were treated by adding an antibiotic solution (ABX) contain-
ing ampicillin (0.1 mg/mL, nal concentration in drinking water),
streptomycin (0.5 mg/mL, nal concentration in drinking water),
and colistin (0.1 mg/mL, nal concentration in drinking water)
(Sigma-Aldrich) to sterile drinking water. Solutions and bottles
were changed 3 times and once weekly, respectively. The antibiotic
activity was conrmed by 16S rRNA gene sequencing. Types of
bacteria with a relative abundance of less than 0.01 are deleted in
the Tg group. The duration of ABX treatment was slightly different
based on the experimental settings. In the context of faecal
microbia transplantation experiments, mice received 3 days of ABX
before undergoing faecal microbia transplantation the next day by
oral gavage using animal feeding needles.
FMT experiments
FMT was performed by thawing faecal material. Then, 200 μLof
the suspension was transferred by oral gavage into each ABX-
pretreated recipient. Twelve-month-old C57 mice were rst
treated with an antibiotic cocktail in drinking water for 3 days,
and then 40 mg of the mixed stool suspended in PBS was inserted
by gavage into each mouse 3 times with a 2-day break in
between. After 3 days, 4.2 µg Aβ140 oligomer was injected into
both sides of the hippocampus. The mice were sacriced 3 days
later and used for different analyses.
Bioinformatics analysis
Principal component analysis (PCA) is a method that simplies the
complexity in high-dimensional data while retaining trends and
Article
14
Cell Research (2019) 0:1 – 17
patterns. PCA reduces data by geometrically projecting them onto
lower dimensions called principal components (PCs), with the goal
of nding the best summary of the data using a limited number of
PCs.
28
Principle coordinate analysis (PCoA) is the modied version
of PCA analysis, in which different distance metrics (e.g., Bray-
Curtis distances) can be used, compared to the Euclidean
distances that is solely used in PCA. Both PCA and PCoA analysis
used in this study were performed at Majorbio I-Sanger Cloud
platform (www.i-sanger.com). For correlational analysis, trend
changes in the abundance of bacteria were correlated with
changes in the frequency of immune cells. The exact correlation
coefcient and the p-value (set to 0.05) were calculated using the
Pearson parametric correlation test using the R package
ggcorrplot. Correlational circus maps were generated using the
R package igraph. Pathway analysis and biological function
enrichment analysis were performed using the Kyoto Encyclope-
dia of Gene Genotype (KEGG). Data were enriched using the R
package DOSE,GO.db,GSEABaseand ClusterProler. Only
pathways with a false discovery rate (FDR) corrected p-value of <
0.05 were represented. Bacterial stream plots were performed
using the R package ggalluvial. The k-means algorithm was used
for clustering different types of immune cells to the cluster with
the nearest mean along the time course using the R package
TCseq(Wu M, Gu L (2019). TCseq: Time course sequencing data
analysis. R package version 1.8.0). A standardized z-score transfor-
mation was applied to convert the fraction values to z-scores
before analysis in all heatmaps. Heatmaps and volcano plots are
also plotted using the R packages. The ROC biomarker
analysis and random forest classication were performed with
MetaboAnalyst 4.0 (https://www.metaboanalyst.ca/). The Mouseac
database used in Supplementary information, Fig. S2 is http://
www.mouseac.org/. Other bioinformatics analyses, including the
barplot representation of gut microbiota abundance at the
family level, lists of signicant changing bacteria, etc. were
conducted using the online platform of the Majorbio I-Sanger
Cloud (www.i-sanger.com).
Flow cytometry
Mice were anesthetized, blood samples were collected into EDTA-
containing tubes, and red blood cells were removed using 1 × red
blood lysis buffer. Before tissue collection, the brains were
perfused with ice-cold PBS to avoid sampling the circulating
blood immune cells, and the brains were removed, chopped into
pieces and dissected according to the introduction of the Adult
Brain Dissociation Kit (Miltenyi, Cat No. 130-107-677) using the
gentleMACS dissociator (Miltenyi Biotec). The brain homogenate
was ltered through a 70-μm cell strainer and centrifuged at
300 × gfor 5 min at 4 °C. The cell pellet was resuspended in cold
PBS buffer and centrifuged again at 300 × gfor 5 min at 4 °C. All
samples were counted and adjusted to a density of 23×10
6
/100
μL, labeled with a Live/Dead kit for 30 min, and then centrifuged
at 500 × gfor 3 min at 4 °C. The cells were resuspended in 100 μL
PBS buffer, blocked with anti-CD16/32 (Biolegend, Cat No. 101320)
for 10 min, and incubated with the antibody according to the
manufacturersprotocols at 4 °C for 30 min. The following
antibodies were used in the FACS analysis: CD45(30-F11)-APC-
Cy7(103116, Biolegend), CD11b(M1/70)-FITC (101205, Biolegend),
CX3CR1(SA011F11)-PE-Dazzle 594 (149014, Biolegend), Siglec-H-
BV421 (566581, BD), F4/80(BM8)-BV421 (123132, Biolegend), CD86
(IT2.2)-PE (305438, Biolegend), CD206(15-2)-APC (321110, Biole-
gend), CD206(15-2)-BV785 (321142, Biolegend), CD11c(N418)-PE-
Cy7 (117318, Biolegend), CD8(53-6.7)-Percp-Cy5.5 (100734,
Biolegend), Ly-6C(HK1.4)-PE-Dazzle 594 (128044, Biolegend), Gr-1
(RB6-8C5)-Percp-Cy5.5 (108428, Biolegend), B220(RA3-6B2)-BV421
(103240, Biolegend), CD19(6D5)-PE (115508, Biolegend), CD49b
(DX5)-PE-Cy7 (108922, Biolegend), CD4(GK1.5)-PE-Cy7 (100422,
Biolegend), CD4(GK1.5)-FITC (100406, Biolegend), CXCR3(CXCR3-
173)-BV421 (126522, Biolegend), CCR4(L291H4)-PE-Cy7 (359410,
Biolegend), CCR6(29-2L17)-APC (129814, Biolegend), CD127
(A019D5)-PE (351304, Biolegend), CD25(3C7)-Percp-Cy5.5
(101912, Biolegend), Live/Dead (423104, Biolegend). Cells were
added to 500 μL PBS buffer, centrifuged at 500 × gfor 3 min at 4 °C
and resuspended in 200 μL running buffer. Relevant negative
control, Fluorescence Minus One (FMO) control and each
uorescence compensation sample were used to adjust uores-
cence compensation and identify the populations of interest. Cells
were acquired on a BD Aria III cytometer, and data were analyzed
using FlowJo software.
Antibody array
The following steps were conducted by RayBiotech (Guangzhou,
China). The brain homogenates (from 20 mg tissue) were
analyzed with a glass slide-based antibody cytokine array
including 200 proteins (RayBiotech, GSM-CAA-4000-1). A 100 μL
sample diluent was added to each well and incubated at room
temperature for 30 min. Then, the buffer was replaced with
another 100 μL of sample and incubated at room temperature for
2 h. The samples were discarded and washed 5 times (5 min
each) with 150 μL of 1 × Wash Buffer I and 2 times (5 min each)
with 150 μLof1×WashBufferIIatroomtemperaturewith
gentle shaking. After that, 80 μLofthedetectionantibody
cocktail were added to each well and incubated at room
temperature for 2 h. The slide was washed 5 times (5 min each)
with 150 μLof1×WashBufferIandthen2timeswith150μLof
1 × Wash Buffer II at room temperature with gentle shaking.
Eighty microliters of Cy3 equivalent dye-conjugated streptavidin
was added to each well and incubated at room temperature for
1 h. After 5 washes (5 min each), the signal was visualized
through a laser scanner. The data were then visualized by a
heatmap diagram (www.metaboanalyst.ca).
Brain section preparation
Mice were transcardially perfused with 0.9% NaCl after deep
anesthesia with pentobarbital (100 mg/kg, i.p.). Brain tissues were
quickly removed, frozen and stored at 70 °C. Serial coronal brain
sections (12 μm thickness) were created using a sliding, freezing
microtome (Leica Microsystems), mounted on slides and dried
overnight in the air at room temperature. Tissue sections were
stored at 80 °C or used immediately.
Laser microdissection and Q-PCR analysis
Frozen mouse brain samples were sectioned and collected on PEN
membrane slides (Leica, 11600288). The hippocampus was
isolated by laser microdissection microscopy (Leica Microsystems,
LMD6). RNA was extracted with a RNeasy Micro Kit (Qiagen, 74004)
and reverse transcribed into cDNA (Takara, PrimeScript RT Master
Mix, RR036A). Q-PCR was performed using the ABI 7500 system via
the SYBR method (Takara, SYBR Premix Ex Taq II). Following
primers were used: Synaptophysin-forward: CAGTTCCGGGTGGT
CAAGG; Synaptophysin-reverse: ACTCTCCG TCTTGTTGGCAC; actin-
forward: GCTCTTTTCCAGCCTTCCTT; actin-reverse: AGGTCTTTACG
GAT GTCAACG.
Statistical analysis
In the behavior test, animals were randomly distributed into
different groups. For two group comparisons, an unpaired two-
tailed Students t-test was applied. For signicantly changing
bacteria lists, we used the online platform of the Majorbio I-Sanger
Cloud to perform Wilcoxon rank-sum test, and the P-value was
based on a two-tailed test with FDR corrected, the signicant level
was set to 0.05, and the 0.95 condence intervals were calculated
through the bootstrap algorithm. For more than two group
comparisons, one-way ANOVA or two-way ANOVA followed by
Dunnetts test was performed. All data with error bar are
represented as mean ± SEM. P< 0.05 was considered statistically
signicant. Most of the data were analyzed in GraphPad Prism. For
Article
15
Cell Research (2019) 0:1 – 17
image quantication, IBA-1-positive, Aβ42-positive and phos-
phorylated Tau-positive cells were analyzed by ImageJ v1.8.0 with
areareadout.
ACKNOWLEDGEMENTS
We thank Muming Poo for helpful comments on the manuscript, Danfeng Zhao for
the preparations of 5XFAD Tg mice. We thank Liang Li for the kind support of the
phenylalanine uptake experiment. This work was supported by the Personalized
Medicines-Molecular Signature-based Drug Discovery and Development, Strategic
Priority Research Program of the Chinese Academy of Sciences (Grant No.
XDA12040101) and the National Science & Technology Major Project Key New
Drug Creation and Manufacturing Programof China (Grant No. 2015ZX09101003).
AUTHOR CONTRIBUTIONS
MYG developed hypotheses, conceived the project, provided supervision, and is
responsible for all data, gures, and text. MYG and JD coordinated the study, XYW
performed gut sample preparation, collected gut microbiome 16S sequencing data,
metagenomics data and untargeted metabolomics data and conducted data analysis.
GQS collected brain and blood samples, performed all FACS experiments, collected
data and conducted analysis; TF collected mouse behavioral data, antibody array
data, targeted metabolites data, conducted analysis and drew the gures; XYW, GQS,
TF, JZ, XH, ZQX and CRG performed further data interpretation and provided
experimental suggestions; XKC, JY, YXG, LFR, GQZ, SYY, WL, CD, and DBY performed
experiments or collected animal samples; SSC performed the animal studies; QLZ,
FFL, JL performed metabolomics experiment and data analysis. SFX, and TW prepared
patient samples and conducted part of the clinical data analysis; HW and HYZ
performed animal experiments in APP/PS1 mice and data analysis. XYW, MYG, JZ
wrote the manuscript.
ADDITIONAL INFORMATION
Supplementary information accompanies this paper at https://doi.org/10.1038/
s41422-019-0216-x.
Competing interests: XYW, GQS, TF, JZ, XKC, JY, SSC, YXG, LFR, GQZ, SYY, WL, CD,
DBY and CRG are full-time employees of Shanghai Green Valley Pharmaceutical Co.,
Ltd. The other authors declare no competing interests.
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In recent years, an extensive body of literature focused on the gut–brain axis and the possible role played by the gut microbiota in modulating brain morphology and function from birth to old age. Gut microbiota has been proposed as a relevant player during the early phases of neurodevelopment, with possible long-standing effects in later life. The reduction in gut microbiota diversity has also become one of the hallmarks of aging, and disturbances in its composition are associated with several (age-related) neurological conditions, including depression, Alzheimer’s disease, and Parkinson’s disease. Several pathways have been evoked for gut microbiota–brain communication, including neural connections (vagus nerve), circulating mediators derived by host-bacteria cometabolism, as well as the influence exerted by gut microbiota on host gut function, metabolism, and immune system. Although the most provoking data emerged from animal studies and despite the huge debate around the possible epiphenomenal nature of those findings, the gut microbiota–brain axis still remains a fascinating target to be exploited to attenuate some of the most burdensome consequences of aging.
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