SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

ALZHEIMER’S DISEASE Copyright © 2022

The Authors, some
Reversal of synapse loss in Alzheimer mouse models by rights reserved;
exclusive licensee
targeting mGluR5 to prevent synaptic tagging by C1Q American Association
for the Advancement
of Science. No claim
Joshua Spurrier1†, LaShae Nicholson1†, Xiaotian T. Fang2†, Austin J. Stoner1†, Takuya Toyonaga2, to original U.S.
Daniel Holden2, Timothy R. Siegert3, William Laird1, Mary Alice Allnutt1, Marius Chiasseu1, Government Works
A. Harrison Brody1, Hideyuki Takahashi1, Sarah Helena Nies1,4, Azucena Pérez-Cañamás1,
Pragalath Sadasivam2, Supum Lee2, Songye Li2, Le Zhang1, Yiyun H. Huang2, Richard E. Carson2,
Zhengxin Cai2*, Stephen M. Strittmatter1*

Microglia-mediated synaptic loss contributes to the development of cognitive impairments in Alzheimer’s disease
(AD). However, the basis for this immune-mediated attack on synapses remains to be elucidated. Treatment with
the metabotropic glutamate receptor 5 (mGluR5) silent allosteric modulator (SAM), BMS-984923, prevents
-amyloid oligomer–induced aberrant synaptic signaling while preserving physiological glutamate response.
Here, we show that oral BMS-984923 effectively occupies brain mGluR5 sites visualized by [18F]FPEB positron
emission tomography (PET) at doses shown to be safe in rodents and nonhuman primates. In aged mouse models
of AD (APPswe/PS1E9 overexpressing transgenic and AppNL-G-F/hMapt double knock-in), SAM treatment fully
restored synaptic density as measured by [18F]SynVesT-1 PET for SV2A and by histology, and the therapeutic
benefit persisted after drug washout. Phospho-TAU accumulation in double knock-in mice was also reduced by
SAM treatment. Single-nuclei transcriptomics demonstrated that SAM treatment in both models normalized expression
patterns to a far greater extent in neurons than glia. Last, treatment prevented synaptic localization of the
complement component C1Q and synaptic engulfment in AD mice. Thus, selective modulation of mGluR5 reversed
neuronal gene expression changes to protect synapses from damage by microglial mediators in rodents.

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INTRODUCTION
Despite the millions of individuals afflicted, there remains no
disease-modifying therapy for Alzheimer’s disease (AD). Evidence
of -amyloid (A) peptide accumulation is required for AD diagnosis
(1); however, other hallmarks are misfolded TAU (MAPT) accumu-
lation, phosphorylation and spreading, and microglial and astroglial
reactivity (2, 3). Genetic evidence from dominant early-onset AD
mutations in amyloid precursor protein (APP) and presenilins,
from Down’s syndrome, and from protective APP alleles are all
consistent with a causative role for A (4). Biomarker studies found
A accumulation 2 decades before symptoms, suggesting an early
role for A in disease pathophysiology (5). However, the temporal
disconnection between A accumulation and symptoms, as well as
the clinical failure of multiple A-lowering therapies have expanded
the focus of AD pathophysiology. Genetic studies implicate microglia
in AD risk and progression. For example, triggering receptor
expressed on myeloid cells 2 (TREM2) signaling has been shown to
exert multiple effects on A-driven pathology (6). In addition,
classic complement cascade components have been directly impli-
cated in phagocytic synaptic removal in AD (7), although the basis
of synaptic selectivity is not defined.
Crucial for clinical AD progression from presymptomatic to mild
cognitive impairment (MCI) to dementia is the loss of synapses (8, 9).
1
Cellular Neuroscience, Neurodegeneration and Repair Program, Departments of
Neurology and Neuroscience, Yale University School of Medicine, New Haven, CT
06510, USA. 2Yale PET Center, Department of Radiology and Biomedical Imaging,
Yale University School of Medicine, New Haven, CT 06510, USA. 3Allyx Therapeutics
Inc., New Haven, CT 06513, USA. 4Graduate School of Cellular and Molecular
Neuroscience, University of Tübingen, Tübingen 72074, Germany.
*Corresponding author. Email: jason.cai@yale.edu (Z.C.); stephen.strittmatter@
yale.edu (S.M.S.)
†These authors contributed equally to this work.
Synapse loss was initially documented ultrastructurally at autopsy,
although it can now be tracked indirectly with fluorodeoxyglucose
positron emission tomography (FDG-PET) or directly by synaptic
vesicle glycoprotein 2A (SV2A) PET (10–12). PET tracers targeting
SV2A, which is ubiquitously expressed in synaptic vesicles, permit
quantification of synaptic density from noninvasive scans, facilitat-
ing tracking of neurodegenerative disease progression and drug
development. PET imaging with [11C]UCB-J previously demonstrated
reduced hippocampal synaptic density of patients with MCI relative
to unimpaired controls (10, 12, 13) and was also able to detect drug
rescue of synapse loss in a mouse amyloidogenic model [APPswe/
PS1∆E9 (14); henceforth, APP/PS1] (15). The second-generation
tracer, [18F]SynVesT-1, with longer half-life, higher resolution,
and greater potential for clinical translation, was able to discern
differences in synaptic density between APP/PS1 mice and control
littermates (16).
With regard to biochemical events at failing AD synapses, cellular
prion protein (PRPC) was identified as a high-affinity receptor for
A oligomers (Ao) in an unbiased genome-wide expression clon-
ing screen (17). PRPC exhibits a unique selectivity for oligomeric A
(17–24). In amyloidogenic AD mouse models, Ao binding to PRPC
contributes to synaptic plasticity impairment, learning and memory
deficits, and synapse loss (17, 25–29). Fyn and Pyk2 (PTK2B) kinases
downstream of Ao and PRPC have been previously linked to TAU
(30–34) and implicated in AD risk (35). A screen for postsynaptic
transmembrane proteins linking Ao and PRPC to intraneuronal
signaling identified neuronal metabotropic glutamate receptor 5
(mGluR5) (23, 36–45). Genetic loss and pharmacological inhibition
studies have demonstrated that reduced mGluR5 activity alleviated
synaptic and memory deficits in multiple amyloidogenic AD mouse
models (25,  36,  37,  40–42,  46–54). However, the molecular and
cellular bases mediating the role of mGluR5 in synaptic loss in AD,

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
including neuroglial interactions and complement-driven synaptic
phagocytosis, remain unclear.
Allosteric mGluR5 modulators have been subdivided into posi-
tive (PAMs), negative (NAMs), and silent (SAMs). PAMs enhance
and NAMs suppress glutamate-induced G protein–mediated Ca2+
mobilization and/or shift glutamate efficacy. Multiple NAMs re-
duce both physiological glutamate signaling and Ao-PRP C–­
dependent synaptic deficits. As a dose-limiting side effect, blockade
of glutamate at mGluR5 with a NAM impaired learning and memory
independently of AD (36, 55–60). We identified a SAM, BMS-984923,
that does not alter basal or glutamate signaling (61–63) but does
inhibit the PRPC-mGluR5 interaction to prevent pathological Ao
signaling (39). This SAM has the potential for expanding the thera-
peutic window for mGluR5 as a disease-modifying AD target.
Here, we use BMS-984923 to explore the connection between
neuronal mGluR5 and glia-dependent synapse loss. We showed
that altered expression of neuronal and glial genes modified neuro-­
immune interaction in murine amyloidogenic (APP/PS1) and AD
{homozygous double knock-in strain [APPNL-G-F/hMAPT (64, 65)];
henceforth labeled dKI} models, resulting in reduced synaptic
density. Treatment with SAM restored synaptic density. Comple-
ment tagging of synapses destined for removal is reported to attract
microglia and astroglia for phagocytosis of synaptic fragments (66).
We showed here that SAM treatment prevented synaptic localiza-
tion of C1Q without altering total C1Q amount or overall gliosis,
suggesting that targeting mGluR5 might be effective for producing
therapeutic effects in AD.
RESULTS
SAM occupies mGluR5 receptors at well-tolerated doses
Our previous data suggested that the mGluR5 SAM, BMS-984923,
is able to reduce AD-related phenotypes in cell culture, brain slices,
and amyloidogenic transgenic (TG) mice (39). Here, to assess
mGluR5 receptor occupancy, we completed [18F]FPEB PET in
rhesus monkeys and in mice. In monkeys, single intravenous doses
of SAM (0.03 to 1.0 mg/kg) blocked mGluR5  in the brain in a
dose-dependent manner (Fig.  1,  A  and  B). Receptor occupancy
from Lassen plots ranged from 27% for the 0.03 mg/kg dose to
94.4 ± 0.4% for the 1.0 mg/kg dose (Fig. 1B). Plasma total SAM
concentrations were measured throughout the course of the scan.
The full dose-response curve revealed that SAM bound to mGluR5
with an ED50 (median effective dose) of 0.11 ± 0.02 mg/kg dose or
with an IC50 (median inhibitory concentration) of 24.8 ± 5.1 ng/ml
total plasma concentration. Separate plasma protein binding studies
indicated a free fraction of 0.1 to 0.5% in different species (data file
S1A); therefore, the measured IC50 value equates to a free drug
concentration of about 0.3 nM, closely similar to the measured
0.6 nM Ki (inhibition constant) for displacement of [3H]MPEPy for
in vitro mGluR5 binding (39). In mice, a single oral dose of SAM
(7.5 mg/kg) administered 2 hours before [18F]FPEB PET blocked
brain mGluR5 with an average receptor occupancy of 98% across
brain regions (Fig. 1C). To further assess occupancy of mGluR5, we
tested the ability of SAM pretreatment to prevent PAM-induced
seizures in mice (39, 67). Doses at 3.75 mg/kg or above completely
blocked mGluR5-PAM–induced seizures (Fig. 1D; ED50 = 1.5 ±
0.8 mg/kg), whereas high doses of the chiral enantiomer of BMS-
984923 (SAM enantiomer, eSAM, at 30 mg/kg) had no effect on
seizures, demonstrating stereoselective receptor binding (Fig. 1D).
Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022
To evaluate the translational potential of SAM, pharmacokinetic,
metabolic, selectivity, and toxicological analyses were pursued.
Studies were conducted in mouse, rat, dog, and monkey to measure
the plasma kinetics of SAM after intravenous and oral administra-
tion. We also assessed the metabolic stability and profile of SAM,
and observed reduced stability in dog, which led us to choose monkey
rather than dog as the second toxicology species (data file S1B).
Metabolite profiling of SAM identified unchanged parent compound
and 38 tentative minor metabolites across human and preclinical
species; metabolic pathways observed in human were also observed
in investigated preclinical species, and no major metabolite com-
prised more than 5% of total (data file S1B). Whereas plasma protein
binding of SAM was >99% in rat, monkey, and human (data file S1A),
oral bioavailability was 50 to 90%, and mouse brain concentrations
were about twice plasma concentrations (data file S1C). Safety evalu-
ation of SAM included a standard Good Laboratory Practice (GLP)
mutagenic program, which demonstrated that SAM was not muta-
genic (data file S1D). In addition, rats tolerated single oral doses of
SAM at 5, 15, and 45 mg/kg without relevant effects on neurobehav-
ioral function, as evaluated using a modified Irwin test, or on body
temperature (data file S1E). Repeat-dose toxicity studies of up to 7 days
in rat and monkey were used to set the dose for the GLP 28-day
repeat-dose studies in both species. Rats received 5, 15, or 45 mg kg−1
day−1, and monkeys received 10, 50, or 200 mg kg−1 day−1. From the
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28-day GLP studies, the no-observed-adverse effect level (NOAEL,
data file S1E) doses were 15 and 200 mg kg−1 day−1 for rat and mon-
key, respectively. The 28-day average maximum concentration and
area under the curve values at the NOAEL are 10,700 ng/ml and
191,000 ng hour/ml in rat and 15,100 ng/ml and 169,000 ng hour/ml
in monkey. The average exposure over 24 hours in monkey at NOAEL
was more than 250-fold greater than the plasma IC50 measured from
the receptor occupancy study using [18F]FPEB PET.
To further evaluate the specificity and selectivity of SAM, in vitro
assays were completed to evaluate any potential interactions with
other targets. SAM was tested in a total of 508 binding and cell-
based functional assays in a CEREP panel using a single concentra-
tion at 10 M in duplicate (data file S1F). Overall, SAM showed
no affinity/potency or a selectivity >300-fold for all targets, with
the exception of PAR1 (protease-activated receptor 1) and PR
(progesterone receptor) for which there was a selectivity between
100- and 300-fold relative to the mGluR5 Ki of 0.6 nM. We assessed
the in vitro potential for SAM to be an inhibitor of the human trans-
porters P-glycoprotein (P-gp), organic ion transporting polypeptide
1B1 (OATP1B1), OATP1B3, and BSEP, as well as the potential of
SAM to be a substrate of the human transporter P-gp. The IC50’s for
each were all far above efficacious SAM concentrations, and SAM
showed no evidence to be a P-gp substrate (data file S1G). Further-
more, we investigated potential CYP inhibition and activation from
SAM parent and metabolite compounds and found that IC50’s were
all far above efficacious SAM drug concentration (data file S1G).
SAM inhibited hERG (human ether-a-go-go related gene) in a GLP
cell culture assay with an IC50 of 1.14 M, 1900-fold above efficacious
exposure concentration (data file S1H). During an in vivo monkey car-
diovascular study at doses up to 200 mg/kg, the QT interval cor-
rected for heart rate was unaltered, and there was no observed effect
on respiratory, central nervous system (CNS), or cardiovascular
function. Thus, oral dosing with SAM occupied brain mGluR5 sites
and was well tolerated at exposures more than 250-fold greater than
required to occupy 50% of the mGluR5 receptor sites.
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

A Rhesus - [18F]FPEB PET B Rhesus: [18F]FPEB receptor occupancy

1 month, followed by 1 month of drug
10.0 ED50: 0.11 + 0.02 mg/kg washout, which is more than 60 half-
9.0
100 IC50: 24.84 + 5.05 ng/ml
lives in mice. After 1 month of treatment,
MRI

8.0
vehicle-treated TG animals exhibited
75 significant (P < 0.05) reduction of both

Receptor occupancy (%)

7.0
the presynaptic marker SV2A and the
6.0
50
RO by plasma conc_M postsynaptic marker postsynaptic den-
Baseline

5.0
RO by plasma conc_F
sity protein 95 (PSD-95), in both den-
RO by dose_M
4.0 25 RO by dose_F
tate gyrus (DG) and CX, compared
3.0
Non-lin. fit by plasma conc. with the WT, whereas synaptic markers
Non-lin. fit by dose in SAM-treated APP/PS1 animals were
0
2.0
restored to WT amounts in all contexts
1.0 mg/kg

0.0 0.2 0.4 0.6 0.8 1.0 1.2

1.0 Dose (mg/kg)
(P  >  0.05), consistent with previously
0.0
SUVRCB 0 50 100 150 200 250 300
published results (fig. S2, A to E) (39).
Plasma conc (ng/ml)
This rescue persisted for at least a
month after treatment cessation, as
C Mouse - [18F]FPEB PET D Mouse: Blockade of PAM-induced seizure SAM-treated TG samples had signifi-
6.0 ED50: 1.5 + 0.8 mg/kg cantly higher (P < 0.01) synaptic density
MR template

1.0
(with both SV2A and PSD-95 in both DG
5.0
and CX) than vehicle-treated TG samples
0.8 after drug washout (Fig. 2, G to K, and
Normalized seizure score

4.0
fig. S2, F to H). Together, the data
0.6
showed a sustained disease-modifying
Vehicle

3.0
0.4
benefit of blocking the PRPC/mGluR5

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SAM
synaptic signaling pathway.
2.0
0.2 eSAM
mGluR5 SAM rescues synapse
density in dKI APPNL-G-F/
7.5 mg/kg

1.0 0.0
0.001 0.01 0.1 1 10
Oral dose (mg/kg) hMAPT mice
0.0
SUVRCB As APP mutant mice can have confound-
ing issues resulting from overexpres-
Fig. 1. SAM receptor occupancy of mGluR5. (A) Template MR images (top) and aligned PET images from a typical sion, competition with endogenous genes,
rhesus brain subject before (middle) and after intravenous dose of SAM (1 mg/kg, bottom), summed from 30 to 45 min
and transgenic promotors, we also tested a
after [18F]FPEB injection. Activity is expressed as SUVR, normalized using cerebellum. (B) Relationship between measured
SAM plasma concentration and mGluR5 receptor occupancy. Fit shown is two-parameter nonlinear mixed effect
homozygous APP NL-G-F/hMAPT dKI
(NLME) model. (C) Template MR images (top) and aligned averaged mouse brain PET images after oral dose of either strain (64, 65). We repeated the mGluR5
vehicle (middle) or SAM (7.5 mg/kg, bottom), summed from 45 to 60 min after injection of [18F]FPEB. Activity is ex- SAM treatment from 12 to 13 months
pressed as SUVR, normalized using cerebellum. (D) Mice were treated with increasing doses of SAM (0 to 7.5 mg/kg) of age in dKI mice and assessed SV2A
or SAM enantiomer (eSAM; 7.5 to 30 mg/kg) by oral gavage 90 min before receiving PAM (20 mg/kg) by intraperitoneal PET and synaptic immunohistology in
injection. Graphs depict the normalized severity of seizure activity (based on Racine scale) after PAM administration. different cohorts (fig. S1, D to G). Base-
line [18F]SynVesT-1 PET found decreased
synapse density in transgenic dKI mice
mGluR5 SAM rescues synapse density in TG compared to WT at 12 months of age (Fig. 3A), which was further
APPswe/PS1∆E9 mice confirmed by region of interest (ROI)–based analysis to be 19% lower
We sought to evaluate whether SAM rescue of preexisting synaptic hippocampal SUVR-1 in dKI mice compared to WT (Fig. 3B,
density deficit in aged AD mice was observed by repeated synaptic P < 0.01). In addition,1-month SAM treatment increased synaptic
PET imaging. Mice were aged to the point that synapse loss was density by 17% as detected by posttreatment [18F]SynVesT-1 PET
detectable using SV2A PET, a tool for monitoring synapse density compared to baseline PET in the same dKI mice (P < 0.05) (Fig. 3, C
with clinical utility in AD and other conditions (fig. S1, A to C and G). and D) to a density comparable to WT mice.
At 12 to 13 months of age, a comparison of baseline [18F]SynVesT-1 We sought to confirm the PET end point by immunohistological
PET found reduced [18F]SynVesT-1 binding in the hippocampus analysis. Both hippocampal and cortical sections of vehicle-treated
(HC) and cerebral cortex (CX) of APP/PS1 relative to wild-type dKI mice exhibited significantly (P < 0.05) reduced SV2A and PSD-
(WT) mice (Fig. 2, A to C; P < 0.0001). Rescans of the same mice 95 punctate staining relative to WT controls (Fig. 3, E to I, and fig.
after a 1-month treatment with the mGluR5 SAM showed a signifi- S3, A to C). In particular, 1-month treatment with SAM restored
cant (P < 0.0001) increase in hippocampal [18F]SynVesT-1 standard- synaptic markers to amounts equal to that of WT mice. To the
ized uptake value ratio minus 1 (SUVR-1) in APP/PS1 compared to extent that the amount of the pre- and postsynaptic markers reflects
WT mice (Fig. 2, D to F). synapse density, then the changes are expected to be similar in
We also tested whether SAM had a chronic disease–modifying individual animals. SV2A and PSD-95 immunoreactivity strongly
effect after treatment cessation by analyzing synaptic marker im- correlated with one another in both DG and CX from both APP/PS1
munostaining. Twelve-month-old APP/PS1 mice were treated for and dKI animal models (fig. S4, A to D; P < 0.0001). Furthermore,

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

Fig. 2. In vivo and in vitro assess- A Baseline SUVRBS-1 B C

ment of synaptic density in APP/ Avg. age = 56 + 5 weeks
Wild type vs. APP/PS1
1.0 ****
PS1 mice. (A) Averaged baseline 1.2 7.0
18 [18
F]SynVesT-1 PET
[ F]SynVesT-1 PET images for WT [ F]SynVesT-1 PET
18

(top) and APP/PS1 (bottom) mouse 1.0 6.0 0.75

brain. Activity is expressed as SUVR-1

Wild type

SUVRHC/BS -1
(normalized to brain stem). (B) Voxel-­ 0.8 5.0
wise analysis t value map compar- 0.5

ing APP/PS1 to WT mouse brain. 0.6 4.0

Color scale represents decrease in
0.25
APP/PS1 compared to WT. (C) ROI- 0.4 3.0

based comparison of hippocampal APP/PS1

[18F]SynVesT-1 SUVR-1 shows synapse 0.2 2.0
0.0
density in APP/PS1 compared to WT WT APP/PS1
0.0 1.0
mice. (D) Voxel-wise analysis t value
map comparing baseline and post- D APP/PS1-SAM E F
treatment synaptic density in APP/ Pre vs. Post
ns ns ns
**** 1.0 ****
1.0
PS1 mice. Color scale represents [ F]SynVesT-1 PET
18 7.0
0.9
increase in posttreatment compared 0.9
0.8
to pretreatment mice. (E) ROI-based 6.0
18 0.7
comparison of hippocampal [ F] 0.8

SUVRHC/BS -1

SUVRHC/BS -1
SynVesT-1 SUVR-1 from aged WT 5.0 0.6
0.7
and APP/PS1 animals at baseline (Pre) 0.5

and after SAM treatment (Post). 4.0 0.4

0.6
(F) Baseline (Pre) and posttreatment 0.3
3.0
(Post) hippocampal [18F]SynVesT-1 0.2
0.5
SUVR-1 from SAM-treated APP/PS1 0.1

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2.0
animals [seventh and eighth bars in 0.0 0.4
(E)] illustrates individual changes. 1.0
Pre Post Pre Post Pre Post Pre Post Pre Post
WT-veh WT-SAM APP/PS1 APP/PS1
(G) Representative images of SV2A veh SAM
immunofluorescent staining of DG
and CX of APP/PS1 mice after a G Post-washout SV2A staining in APP/PS1 H I
Dentate gyrus Cortex 10 10
month-long treatment washout ***ns **** ****ns ****
period. Scale bar, 20 m. Images from
8 8
SV2A staining in DG and CX of WT
DG SV2A+ area (%)

CX SV2A+ area (%)

animals and from PSD-95 in DG of
6 6
WT and APP/PS1 animals are shown
in fig. S2 (F to H). (H to K) Fractional
Vehicle

4 4
area of SV2A (H and I) or PSD-95
(J and K) immunoreactive puncta 2 2
from DG (H and J) or CX (I and K) of
post-washout animals treated with 0 0

vehicle or SAM. In (C), (E), (F), and 20 µm

WT-veh WT-SAM APP/PS1-veh APP/PS1-SAM
(H) to (K), each individual mouse is
shown as a single dot. In (C), data J K
6 6
are presented as means ± SEM from ** ns ** ** ns ***
n = 24 mice per group and compared
by unpaired t test. In (E), data are
CX PSD-95+ area (%)
DG PSD-95+ area (%)

presented as means ± SEM from n = 12 4 4

mice per group and compared by

SAM

repeated-measures two-way ANOVA

with Holm-Šídák’s multiple compari- 2 2

sons test. In (H) to (K), data are pre-

sented as means ± SEM from n = 14
to 24 mice per group and compared 0 0
by one-way ANOVA with Holm-Šídák’s
multiple comparisons test. **P < 0.01,
***P < 0.001, ****P < 0.0001. ns, not significant.

SV2A staining in DG was significantly correlated with hippocampal
SV2A PET SUVR-1 (fig. S4E; P < 0.0001), providing cross-validation
of the two methods.
Multiple studies have provided clinical and preclinical evidence
supporting an increased incidence of AD in females compared to
males. One recent experiment investigating sex differences in AD con-
cluded that the mGluR5 NAM, CTEP, blocked A pathophysiology
and improved cognitive declines selectively in male but not female
APP/PS1 mice (54). In contrast, our SV2A PET studies showed
rescue of synaptic density loss in females by treatment with SAM in

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

Fig. 3. In vivo and in vitro as- A B C D

WT vs dKI dKI-SAM
sessment of synaptic density ** 1.0
0
Avg. age = 56 + 3 weeks 0
1.0 Pre vs. Post
*
in dKI mice. (A) Voxel-wise 18
[ F]SynVesT-1 PET 7.0 18
[ F]SynVesT-1 PET 7.0 0.9
9
analysis t value map comparing
synaptic density of dKI to WT 6.0
0.8
8
6.0
mice. Color scale represents de-

SUVRHC/BS -1
SUVRHC/BS -1
0.7
7
crease in dKI compared to WT. 5.0 5.0
0.6
6
(B) ROI-based comparison of 0.5
5

hippocampal [ 18 F]SynVesT-1 4.0 4.0 0.5

5
SUVR-1 in dKI mice compared to
0.4
4
WT (WT values are from APP/PS1 3.0 3.0
cohort presented in Fig. 2C). 3
0.3

(C) Voxel-wise analysis t value 2.0 0

0.0 2.0
2
0.2
map comparing synaptic densi- WT dKI Pre Post
ty pre- and posttreatment with 1.0 1.0
SAM. Color scale represents in-
crease in posttreatment com- E Dentate gyrus PSD-95 staining F G
WT dKI
pared to pretreatment mice. 5 **** ns
**** 4 ****ns ****
(D) Paired comparison of ROI-
based analysis of hippocampal 4

CX PSD-95+ area (%)

DG PSD-95+ area (%)
3
SUVR-1 between pretreatment
and posttreatment dKI mice. 3
2
(E) Representative images of
Vehicle

2
PSD-95 immunofluorescent stain-
ing of DG of WT and dKI mice 1
1
after treatment with vehicle or
SAM. Scale bar, 20 m. Images

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0 0
from SV2A staining of DG and 20 µm
WT-veh WT-SAM dKI-veh dKI-SAM
CX and from PSD-95 staining of
CX are shown in fig. S3 (A to C). H I
(F to I) Fractional area of PSD-95 10 * ns 0.063
6 ** ns *
(F and G) or SV2A (H and I) im-
munoreactive puncta from DG 8

CX SV2A+ area (%)

(F and H) or CX (G and I) of WT DG SV2A+ area (%)
4
and dKI animals treated with 6
SAM

vehicle or SAM. (J) Representa-

tive images of SV2A and PSD-95 4
2
immunofluorescent costaining
2
of CA1 of dKI mice after treat-
ment with vehicle or SAM. Scale
0 0
bar, 2 m. (K) Quantification of
synaptic loci (per cubic micrometer)
J CA1 SV2A/PSD-95 staining K
in CA1 of vehicle- or SAM-treated dKI-veh dKI-SAM 200
dKI animals. In (B), (D), (F) to (I), *
and (K), each individual mouse is
Synaptic loci (% dKI-veh)

shown as single dot. In (B), data 150

are presented as means ± SEM

from n = 24 (WT) or 10 (dKI) 100
mice per group and compared
by unpaired two-tailed t test. In
(D), data are compared by paired 50

two-tailed t test. In (F) to (I), data

are presented as means ± SEM 0
from n = 21 to 34 mice per group 2 µm dKI-veh
and compared by one-way ANOVA dKI-SAM
with Holm-Šídák’s multiple com-
parisons test. In (K), data are
presented as means ± SEM from n = 12 (dKI-veh) or 11 (dKI-SAM) mice per group and compared by unpaired t test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns, not significant.

both the APP/PS1 and dKI models of AD. To further investigate a
potential sex difference, we carried out a meta-analysis of our SV2A
and PSD-95 histology data segregated by sex (fig. S5, A to C). Stain-
ing results from both synaptic markers in both DG and CX were
compiled into a synaptic score for each individual animal (see
Materials and Methods). In the APP/PS1 cohorts (fig. S5A), the dKI
cohorts (fig. S5B), and in an analysis of the two models combined
(fig. S5C), both males and females were fully rescued by SAM treat-
ment with highly robust statistical significance (AD-veh versus
WT-veh: P < 0.01; AD-SAM versus WT-veh: P > 0.05).

Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022 5 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
To further confirm that these synaptic markers reflected synap-
tic density, we assessed the juxtaposition of SV2A and PSD-95
puncta in three dimensions using a modified SEQUIN method (68).
For this purpose, we used super-resolution Airyscan microscopy in
confocal Z series of double-stained sections of the CA1 from vehicle-
and SAM-treated dKI mice and scored synapses as pairs of puncta
stained with pre- and postsynaptic proteins within 250 nm of one
another. We observed that SAM treatment significantly (P < 0.05)
increased the density of synaptic loci relative to vehicle-treated dKI
(Fig. 3, J and K). Thus, in multiple strains with multiple measures,
blocking the mGluR5 pathway with SAM had a disease-modifying
effect to support recovery of synaptic density.
We considered the extent to which our primary outcome,
synapse density, matched with behavioral deficits in these mouse
models. We have previously shown that recovery of synapse density
associated with behavioral benefit after treatment with SAM in
APP/PS1 mice (39). SAM rescued spatial memory in the Morris
water maze, novel object memory, and passive avoidance learning.
We also showed that rescue of APP/PS1 synapse density by the
related mGluR5 NAM compound, MTEP, is accompanied by
recovery of spatial memory and novel object memory (19, 27, 36).
Here, we measured memory performance in the dKI model. At
3 months of age, the dKI mice exhibited no spatial memory deficit
(fig. S6, A and B), but 12-month-old dKI mice treated with vehicle
for 1 month exhibited significantly impaired learning (fig. S6C;
P < 0.0001) and could not recall a hidden platform location during
a probe trial (fig. S6D). In contrast, age-matched dKI mice treated
with SAM for 1 month spent significantly (P = 0.0059) more time in
the target quadrant than adjacent areas (fig. S6C). These memory
phenotypes occurred without any effect of dKI genotype or SAM
treatment on the ability to swim to a visual platform (fig. S6E). DG
SV2A area was also positively correlated with spatial memory per-
formance (fig. S4F; P = 0.048). Thus, the ability of SAM compound
treatment to restore synapse density in both APP/PS1 and dKI mice
correlated with recovery of spatial memory.
Phospho-TAU accumulation is reduced in dKI mice by
mGluR5 SAM
TAU hyperphosphorylation and neurofibrillary tangles are key
components of AD pathology and are thought to be driven by
upstream A synaptopathology in human brain and to synergize with
A for further synapse loss (69–71). However, many mouse
amyloidogenic AD models (including APP/PS1) show little or no
TAU pathology. The dKI strain inserts human MAPT into the
mouse Mapt locus and shows increases in certain phospho-TAU
(pTAU) epitopes without tangles (65). Therefore, we assessed pTAU
pathology in the dKI strain and its response to SAM treatment. The
dKI mice were previously shown to have higher AT8 immuno-
reactivity in cortical brain tissue (65). Here, we confirmed by im-
munohistochemistry and biochemistry that AT8 amounts were
increased in cortical tissue from 12-month-old dKI animals
(Fig. 4, A to D; P < 0.0001). One-month SAM treatment significantly
reduced AT8 in the CX (Fig. 4, C and D; P < 0.05).
We evaluated two additional disease-associated pTAU markers
in dKI brain sections. TAU phosphorylation at S396/S404 has been
reported as an early event in AD progression (72), and the pT217-
TAU epitope has been documented as a specific plasma and
cerebrospinal fluid (CSF) marker of AD tauopathy (73). pS396-TAU–­
positive area was significantly (P < 0.0001) increased in dKI cerebral
Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022
CX and in CA1, and this signal was decreased significantly (P < 0.01)
in CX by 1-month SAM treatment (Fig. 4, E to G). pT217-TAU–
positive area, although less than pS396-TAU, still showed accu-
mulation (P  <  0.0001) in cerebral CX and in CA1, and was
significantly decreased (P < 0.05) by SAM treatment in both
regions (Fig. 4, H and I). Thus, rescue of synapse loss by SAM in
the dKI model might, at least in part, be mediated by reduced pTAU
accumulation; alternatively, reduced pTAU accumulation may be
secondary, in part, to synapse recovery with SAM.
Single-nuclei transcriptomics of mGluR5 SAM–treated mice
reveals neuronal rescue
To explore the molecular underpinnings linking SAM treatment
with synaptic recovery in an unbiased manner, we conducted deep
single-nuclei transcriptomic analysis of amyloidogenic and AD
models with and without mGluR5 SAM treatment (Figs. 5 and 6 and
figs. S7 to S13). We collected brain tissue from aged WT and APP/PS1
TG mice treated twice daily with either vehicle (V) or SAM (3.75 mg/kg)
(D) for 1 month from 12 to 13 months of age (WTV, WTD, TGV,
and TGD), as well as a separate cohort of similarly aged and treated
WT and dKI animals (WTV, WTD, dKIV, and dKID) (Fig. 5A).
Each group included four mice, and all were on a pure C57/Bl6J
background. Cerebral CX and HC from each brain were dissected,
and the two regions were processed together for each mouse.
Downloaded from https://www.science.org at Yale University on June 14, 2022
Nuclei isolated by sucrose density gradient centrifugation were pro-
cessed for mRNA profiles with the 10X Genomics platform (~15,000
per sample). In total, nearly 500,000 nuclei were sequenced to a
depth of ~20,000 reads each (fig. S7, A to D). The resulting DNA
sequences were processed in Seurat, and no confounding batch
effects were observed (fig. S8, A and B). Integrated data were
clustered in Uniform Manifold Approximation and Projection (UMAP)
space (fig. S8C). Cell clusters were identified by canonical cell type
markers, with at least three for each cell type (fig. S8D). The expres-
sion of one marker for each is shown (fig. S8G). Both models
exhibited similar numbers and proportions of each cell type (fig.
S8, E and F).
We examined expression profiles for AD-associated and SAM-­
regulated differentially expressed genes (DEGs) across cell types.
All cell types showed substantial changes induced by APP/PS1 or
dKI compared to WT. For excitatory neurons (ExNs), a “volcano”
plot revealed the pattern of up- and down-regulated genes in TGV
or dKIV group compared to the WTV group (Fig. 5, B and D).
In contrast, many fewer DEGs were present in the comparison of
SAM-treated TG or dKI samples with the WTV group (Fig. 5, C and E).
Indeed, 83 and 63% of AD-associated expression changes within
ExNs of APP/PS1 and dKI samples, respectively, were corrected by
SAM treatment (Fig. 5F). Expression patterns across the different
groups for each DEG revealed that SAM treatment restored both
up-regulated and down-regulated genes (Fig.  5F). Similar rescue
was observed in inhibitory neurons (InNs) (Fig. 5G). The list of all
SAM-corrected DEGs from ExNs and InNs and from either mouse
model was analyzed for pathway enrichment with ClueGO (74).
Differences in enrichment of networks composed of Gene Ontology
(GO) cell compartment terms were detected (fig. S9, A to C).
Specifically, neuronal SAM–corrected DEGs were strongly enriched
in GO terms related to neuronal synapses, with less prominently
populated networks related to ribosome, V–adenosine triphospha-
tase (ATPase), and glial projection. This analysis is consistent with
expected primary action of SAM and mGluR5 at neuronal synapses.
6 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

A Medial cortex AT8/NeuN staining B WT-veh WT-SAM dKI-veh dKI-SAM

WT dKI 75 kDa

50 kDa
AT8

50 kDa
Vehicle

37 kDa Actin

C D
0.20 ****ns ** 50 **** *
50 m
*
40

Medial cortex AT8+ area (%)

0.15

Cortical AT8/actin
30
0.10

20
SAM

0.05
10

0.00 0

Downloaded from https://www.science.org at Yale University on June 14, 2022

WT-veh WT-SAM dKI-veh dKI-SAM

E F H
Medial cortex pS396 staining
1.5 **** ** **** *
dKI
** 0.03
*
WT

Medial cortex pT217+ area (%)

Medial cortex pS396+ area (%)

1.0 0.02
Vehicle

0.5 0.01

0.0 0.00
100 m
G I
0.25 **** 0.086
0.025 **** *
** ****
0.20 0.020
CA1 pS396+ area (%)

CA1 pT217+ area (%)

0.15 0.015
SAM

0.10 0.010

0.05 0.005

0.00 0.000

Fig. 4. In vitro assessment of TAU pathophysiology in dKI mice. (A) Representative images of AT8 and NEUN immunofluorescent staining of medial CX of WT and dKI
mice after treatment with vehicle or SAM. Scale bar, 50 m. (B) Representative immunoblots of AT8 amounts in cortical tris-buffered saline (TBS) fraction of WT and dKI
animals treated with vehicle or SAM; actin was used as loading control. (C) Fractional area of AT8 immunoreactive puncta from medial CX of treated WT and dKI animals,
from micrographs as in (A). (D) Quantification of AT8 summed from TBS and Triton X-100 fractions, normalized to actin, from immunoblots as in (B). (E) Representative
images of pS396 immunofluorescent staining of medial CX of WT and dKI mice after treatment with vehicle or SAM. Scale bar, 100 m. (F and G) Fractional area of pS396
immunoreactive puncta from medial CX (F) or hippocampal CA1 (G) of WT and dKI animals treated with vehicle or SAM. (H and I) Fractional area of pT217 immunoreactive
puncta from medial CX (H) or hippocampal CA1 (I) of WT and dKI animals treated with vehicle or SAM. In (C) and (D) and (F) to (I), each individual mouse is shown as single
dot. Data are presented as means ± SEM from n = 7 to 20 mice per group and compared by one-way ANOVA with Holm-Šídák’s multiple comparisons test. *P < 0.05,
**P < 0.01, ****P < 0.0001. ns, not significant.

Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022 7 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

Fig. 5. snRNA-seq shows SAM-mediated A 4 WT veh 4 WT veh 12 months age 10X Genomics process & sequence
correction of expression changes in neu- 4 WT SAM 4 WT SAM Treat for 1 month 482,680 total nuclei sequences
4 APP/PS1 veh 4 dKI veh Cortex + hippocampus Mean UMI per nucleus ~1800
ronal and glia cell populations from 4 APP/PS1 SAM 4 dKI SAM Isolate nuclei Dataset (UMI x Nuc) = 8.7 × 10E08
amyloidogenic and AD mouse models.
(A) Cerebral CX and HC from WT, APP/PS1, B APP/PS1 excitatory neurons D dKI excitatory neurons
and dKI mice treated with vehicle or SAM 1000 Camk2n1
Hspa8 1000 Camk2n1Cst3 Hspa8

−Log10 (P value)
500 Ubb Tmsb4x Atp1b1

−Log10 (P value)
Itm2b Cd47 Calm1 Eef1a1 500 Actb
(n = 4 mice per group) from 12 to 13 months 100
Negr1
Aldoa Fth1 Ptgds 100 Calm1
Aldoa Fth1
Hsp90ab1

of age were fractionated to single nuclei and AD-veh Atp1b1

Hsp90ab1
Ubb Itm2b Pgam1 Slc25a4
Mdh1 Tubb2a Ptgds Arl6ip1 Negr1
vs Tmsb4x Mdh1
processed by 10X Genomics. (B to E) Volca- WT-veh
10
Slc2a13 Pgam1 Cst3 Slc25a4
Actb 10
Eef1a1

no plots of DEGs identified in APP/PS1 (B and 1

Arl6ip1
1
Tubb2a Cd47 Slc2a13

−0.6 −0.3 0.0 0.3 0.6 −0.6 −0.3 0.0 0.3 0.6
C) or dKI (D and E) ExN cell populations. Plots Tg-veh vs WT-veh (logFC) dKI-veh vs WT-veh (logFC)
show statistical significance (−log10, P value) C 1000 E 1000

−Log10 (P value)
Hsp90ab1 Camk2n1 Ubb Mdh1 Hspa8 Camk2n1
500
versus magnitude of gene expression chang-

−Log10 (P value)
Calm1 Atp1b1 Cd47 500 Eef1a1
Eef1a1 Tubb2a Slc25a4
100 Slc25a4 Mdh1 Itm2b
es (logFC) of AD-veh (B and D) or AD-SAM (C AD-SAM Aldoa Itm2b
Actb
Hspa8
Arl6ip1
100 Aldoa
Cd47
Calm1
Arl6ip1 Tmsb4x
Fth1 Ubb Pgam1
and E) when compared to WT-vehicle–treated vs
WT-veh
10 Slc2a13 Ptgds
Tubb2a
10 Cst3 Slc2a13
Hsp90ab1 Fth1
mice; vertical dashed line indicates 0.0 logFC, Cst3 Negr1 Pgam1
Tmsb4x Atp1b1
Ptgds Actb
1 1
and vertical solid lines indicate +0.1 logFC. A −0.6 −0.3 0.0 0.3 0.6 −0.6 −0.3 0.0 0.3 0.6
Tg-SAM vs WT-veh (logFC) dKI-SAM vs WT-veh (logFC)
selection of significant, AD-associated DEGs F G
is marked in red, with gene names identified APP/PS1 dKI APP/PS1 dKI
(B and D). DEGs are deemed significant if AD DEGs =127 AD DEGs = 284 AD DEGs = 102 AD DEGs = 274
Not cor. Not cor. Not cor. Not cor.
they exhibit an absolute logFC > 0.1 with SAM cor. 17% SAM cor. SAM cor.
7%
SAM cor.
P < 0.005 (Wilcoxon rank sum test). The 37% 45%
same AD-­associated DEGs are marked in 83% 93%
63% 55%
green (B and D) if their absolute logFC < 0.1.
For full DEG list from all cell types, refer to SAM- SAM-
data file S2. (F to I) Total number of AD-­ corrected P = 2.3e-50 P = 2.5e-73 corrected P = 4.2e-50 P = 4.5e-59
Excitatory neurons

Inhibitory neurons
P value P value
associated DEGs in ExNs (F), InNs (G), microglia

Downloaded from https://www.science.org at Yale University on June 14, 2022

WT-SAM

WT-SAM

WT-SAM

WT-SAM
dKI-SAM

dKI-SAM
(H), and reactive astrocytes (I) from APP/PS1
Tg-SAM

Tg-SAM
WT-veh

WT-veh

WT-veh

WT-veh
dKI-veh

dKI-veh
Tg-veh

Tg-veh
or dKI samples. Pie charts illustrate percent-
age of DEGs that are fully corrected by SAM
2

2
treatment (Fisher’s exact test); the size of the
Z-score scaled exp.

Z-score scaled exp.

1

1
chart is relative to total number of DEGs.
Heatmaps show single DEG expression with-
0

0
in each cell type. Analyses of remaining cell
−1

types are included in fig. S11. −1

−2

−2

H I
APP/PS1 dKI APP/PS1 dKI
AD DEGs = 108 AD DEGs = 390 AD DEGs = 260 AD DEGs = 310
Not cor. Not cor. Not cor. Not cor.
SAM cor. SAM cor. 7% SAM cor. SAM cor.
10%
35% 28%

90% 93% 65% 72%

SAM- SAM-
corrected corrected
SAM-mediated correction was
Reactive astrocytes

P = 7.5e-4 P = 9.4e-9 P = 5.4e-32 P = 4.7e-30

P value P value
predominantly neuronal, because SAM
Microglia

WT-SAM

WT-SAM

dKI-SAM
WT-SAM

WT-SAM

dKI-SAM

Tg-SAM
Tg-SAM

WT-veh

WT-veh

dKI-veh
WT-veh

WT-veh

dKI-veh

treatment failed to correct most DEGs

Tg-veh
Tg-veh

in each glia cell population analyzed

(Fig. 5, H and I, and fig. S10, A to C).
2

2
Z-score scaled exp.

Z-score scaled exp.

Increasing the threshold stringency

1

for differential expression analysis

0

had minimal effect on the propor-

−1

−1

tion of SAM-corrected DEGs across

−2

−2

cell types (fig. S11), highlighting not

only that SAM-mediated correc-
tions were primarily neuronal but
also that treatment corrected at least 73% of neuronal DEGs with in TGD and WTV, indicating that there is complete rescue. Points
the greatest fold-change difference, regardless of model. along the line y = 0 reflect an AD-related phenotype unaffected by
To visualize differential expression transcriptome-wide between SAM treatment. Although the linear regression of all genes showed
TGV, TGD, and WTV, the log fold change (logFC) for WTV com- a highly significant difference from slope  =  0  in every cell type
pared to TGV (AD effect, x axis) was plotted against that for TGD examined (P < 2.2 × 10–16), the effect was much more pronounced in
compared to TGV (SAM effect, y axis) (Fig. 6, A to I, and fig. S12, A neuronal subtypes. For APP/PS1 ExN, the slope was 0.70 and Pearson
to F). In this plot, points along the identity line of slope = 1 are equal R was 0.72, with close to full normalization by SAM (Fig. 6B).

Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022 8 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

A The regression of expression profiles for DEGs in InNs showed a

AD-SAM vs AD-veh (LogFC)
0.6 Slope = 1
Full rescue APP / PS1 DEG
Corrected by SAM slope nearly as close to 1. In these neuronal populations, most DEGs
0.3
Slope = 0 dKI DEG were corrected by SAM [colored pink (APP/PS1) or green (dKI)]
No rescue Corrected by SAM
0.0 and largely fell along the y = x identity line. In contrast, although the
DEG not corrected
by SAM slopes in microglia and astrocytes were also significantly different
−0.3
Transcripts below
from 0 (P < 2.2 × 10−16), they were substantially shallower than in
−0.6 DEG cutoff neurons, because most DEGs failed to be corrected (black dots)
−0.6 −0.3 0.0 0.3 0.6 (Fig. 6, B to I, and fig. S12, A to F).
WT−veh vs AD−veh (LogFC)
Both the APP/PS1 and dKI models showed a substantial (P < 2.3 ×
B APP / PS1
C dKI 10−50) rescue of expression patterns by SAM, in correlation with
0.6
dKI-SAM vs. dKI-veh (LogFC)
0.6 synapse rescue by immunohistology and PET imaging (Figs. 2 and
Tg-SAM vs. Tg-veh (LogFC)

3). We compared the AD-related DEGs of all major cell types of the
Excitatory neurons

0.3 0.3
two models and found significant overlap in each context (P < 4.8 ×
0.0 0.0 10−4) (fig. S13). In ExN, the overlap of DEGs contained 46 genes
−0.3 −0.3
(fig. S10, A and B). Of these 46 core DEGs, most were expression-­
R =0.72 R =0.44 normalized by SAM in both models; 36 were rescued in APP/PS1,
P <2.2e-16 P <2.2e-16
−0.6 −0.6 and 31 in dKI (fig. S13B). Thus, single-nuclei RNA sequencing
−0.6 −0.3 0.0 0.3 0.6 −0.6 −0.3 0.0 0.3 0.6
WT−veh vs. Tg−veh (LogFC) WT−veh vs. dKI−veh (LogFC) (snRNA-seq) profiling methods revealed robust expression changes
D E across multiple cell types and pronounced global rescue by SAM
treatment, but the rescue was far more complete in neurons than
dKI-SAM vs. dKI-veh (LogFC)
Tg-SAM vs. Tg-veh (LogFC)

0.6 0.6
in glia.
Inhibitory neurons

0.3 0.3
Research into the role of glial cells in AD progression has identi-
0.0 0.0 fied expression signatures of disease-associated microglia (DAM)
(75) and astrocytes (DAA) (76) in the 5XFAD TG amyloidogenic
−0.3 −0.3

Downloaded from https://www.science.org at Yale University on June 14, 2022

R =0.61 R =0.46 mouse model, each with unique transcriptomic profiles. Our data
−0.6 P <2.2e-16 −0.6 P <2.2e-16 here demonstrated conservation of a subset of these characteristic
−0.6 −0.3 0.0 0.3 0.6 −0.6 −0.3 0.0 0.3 0.6 DEGs in both cell types (fig. S13, G to I). Although microglia from
WT−veh vs. Tg−veh (LogFC) WT−veh vs. dKI−veh (LogFC)
dKI animals had a higher number of DEGs relative to WT controls
F G
than did APP/PS1 (and thus greater overlap with DAMs), both mouse
dKI-SAM vs. dKI-veh (LogFC)

0.6 0.6
Tg-SAM vs. Tg-veh (LogFC)

models had a similar number of overlapping DEGs with DAA.

0.3 0.3
Microglia

0.0 0.0 C1Q localization to PSD-95 puncta is prevented by mGluR5

blockade, and synaptic engulfment is reduced
−0.3 −0.3
R =0.43 R =0.34
Although our single-nuclei transcriptomic data revealed minimal
−0.6 P <2.2e-16 −0.6 P <2.2e-16 glial normalization by SAM, activation of glial cells and C1Q synaptic
−0.6 −0.3 0.0 0.3 0.6 −0.6 −0.3 0.0 0.3 0.6 tagging have been strongly implicated in AD synapse loss (7, 77, 78).
WT−veh vs. Tg−veh (LogFC) WT−veh vs. dKI−veh (LogFC)
We hypothesized that gliosis and complement release may still
H I occur in the presence of SAM but that innate immune mediators
dKI-SAM vs. dKI-veh (LogFC)

0.6 0.6
Tg-SAM vs. Tg-veh (LogFC)

might fail to target the expression-normalized neuronal synaptic

Reactive astrocytes

0.3 0.3 elements. Specifically, the synapse-rescuing effect of SAM treat-

0.0
−0.3
R =0.37
−0.6 P <2.2e-16
−0.6 −0.3 0.0 0.3 0.6
WT−veh vs. Tg−veh (LogFC)
Fig. 6. Transcriptome-wide correction by SAM treatment in neuronal and glial
cell types. (A) Cell type–specific comparison of AD-associated DEGs and SAM-­
corrected DEGs in APP/PS1 and dKI samples. LogFC between WT-veh and either
APP/PS1-veh or dKI-veh (AD effect) is plotted along the x axis. LogFC between
vehicle- and SAM-treated APP/PS1 or dKI samples (SAM effect) is plotted along the
y axis. Black points represent genes with logFC > 0.1 and P < 0.005. Colored points
(pink = APP/PS1, green = dKI) represent DEGs that were corrected by SAM treat-
ment. Points along the identity line (x = y) represent genes with equivalent differ-
ential expression between AD-SAM and WT-veh, relative to AD-veh, indicating
complete rescue by SAM. Points along the line “y = 0” reflect genes unaffected by
SAM. The regression line (Pearson’s correlations) represents transcriptome-wide
effects of SAM treatment. Expression pattern for different cell types is provided for
ExNs (B and C), InNs (D and E), microglia (F and G), and reactive astrocytes (H and
I). Analyses of remaining cell types are included in fig. S12.
0.0
ment might relate to a prevention of complement tagging of synapses
rather than an upstream reduction of microgliosis or complement
−0.3 production. First, we examined whether the synapse-rescuing
R =0.11
−0.6 P <2.2e-16 mGluR5 SAM normalized gliosis, C1Q amounts, or colocalization
−0.6 −0.3 0.0 0.3 0.6 with PSD-95. We have previously examined astrocyte, microglia,
WT−veh vs. dKI−veh (LogFC)
and A area in the hippocampi of treated and nontreated APP/PS1
mice and found that SAM treatment had no effect on gliosis or
plaque load (39,  79,  80). Here, we obtained similar results for
SAM-treated dKI (fig. S14, A to F). IBA1 (a microglial marker; fig.
S14, A and B), GFAP (an astrocyte marker; fig. S14, C and D), and
thioflavin S (amyloid plaques; fig. S14, E and F) staining were
significantly increased (P < 0.01, P < 0.05, and P < 0.0001, respec-
tively) in 12-month-old vehicle-treated dKI animals, relative to
WT, and none was affected by SAM treatment. To assess total C1Q
protein, we completed C1Q immunoblots in the dKI and APP/PS1
samples with and without SAM treatment. Total C1Q in dKI HC sam-
ples was substantially increased in the vehicle-treated samples rela-
tive to WT, and this was not altered by the SAM treatment (dKI;
Fig. 7, A and B; P < 0.0001).

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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

A B 10.0 **** ns
WT-veh WT-SAM dKI-veh dKI-SAM
****
37 kDa 8.0
C1q WT-veh
25 kDa
6.0

C1q / actin
WT-SAM

50 kDa 4.0 dKI-veh

Actin
37 kDa dKI-SAM
2.0

0.0
C C1q / PSD-95 staining in dentate gyrus D
WT dKI
0.15
****ns **

area overlapping C1q

DG PSD-95+ fractional
WT-veh
0.10

WT-SAM
Vehicle

dKI-veh
0.05
dKI-SAM

5 µm
0.00

Downloaded from https://www.science.org at Yale University on June 14, 2022

E
0.06 ** ns *
area overlapping C1q
DG PSD-95+ fractional

WT-veh
0.04
WT-SAM
SAM

APP/PS1-veh
0.02
APP/PS1-SAM

0.00

F SV2a / GFAP staining in CA1 G

dKI-veh dKI-SAM
0.4
**** *
****
0.3 WT-veh
CA1 SV2a+ area (%)
engulfed in GFAP

WT-SAM
0.2
dKI-veh

dKI-SAM
0.1

5 µm 0.0

Fig. 7. Synaptic localization of C1Q in amyloidogenic and AD mouse models. (A) Total C1Q in HC from WT and dKI animals treated with vehicle or SAM were mea-
sured by Western blotting; actin was used as loading control. (B) Quantification of immunoblot in (A). C1Q intensity was normalized to actin. Data are means ± SEM
from n = 6 mice per group and compared by one-way ANOVA with Holm-Šídák’s multiple comparisons test. (C) Representative images of C1Q and PSD-95 immunofluo-
rescent staining of DG of WT and dKI mice after treatment with vehicle or SAM imaged with Airyscan super-resolution. Scale bar, 5 m. Yellow arrows highlight C1Q/
PSD-95 colocalization. (D and E) Fractional area of PSD-95 immunoreactive puncta overlapping C1Q in DG of dKI (D) or APP/PS1 (E) animals treated with vehicle or
SAM. Each individual mouse is shown as a single dot. Data are presented after measurement by Mander’s coefficient as means ± SEM from n = 6 to 10 mice per group and
compared by one-way ANOVA with Holm-Šídák’s multiple comparisons test. (F) Representative orthogonal views from different planes (x/y, x/z, or y/z) of SV2A and GFAP
immunofluorescent staining of CA1 of dKI mice after treatment with vehicle or SAM. Scale bar, 5 m. (G) Fractional area of SV2A immunoreactive puncta engulfed in GFAP
in CA1 of WT or dKI animals treated with vehicle or SAM. Each individual mouse is shown as a single dot. Data are presented as means ± SEM from n = 8 to 10 mice per
group and compared by one-way ANOVA with Holm-Šídák’s multiple comparisons test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022 10 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

To assess innate immune tagging of synapses by complement,
we analyzed DG sections by super-resolution Airyscan microscopy
with antibodies against C1Q and PSD-95 (Fig. 7C). At 12 months
old, the fraction of PSD-95 overlapping with C1Q was significantly
increased in both dKI (Fig. 7D; P < 0.0001) and APP/PS1 (Fig. 7E;
P < 0.01) vehicle-treated mice, consistent with published reports in
other models (7, 81, 82). In contrast, animals treated for 1 month
with SAM exhibited PSD-95/C1Q colocalization that was reduced
relative to vehicle-treated animals (P < 0.05) and was indistinguishableis essential for early diagnosis of AD. SV2A, a ubiquitously and
from WT. C1Q overlap with the presynaptic marker SYNAPSIN
1/2 was also significantly elevated (P < 0.05) in DG of vehicle-treated throughout the brain. Expanding on recent studies (16, 84, 85), we
dKI mice and restored to WT amount in SAM-treated animals
(fig. S15, A and B). This finding is consistent with the hypothesis
that SAM normalization of synaptic signaling and neuronal gene
expression in these amyloidogenic and AD models eliminates syn-
aptic tagging by complement, despite persistent glial activation and
C1Q production.
After synaptic tagging by C1Q, both astrocytes and microglia are
thought to participate in synaptic removal by phagocytic mecha-
nisms. One study highlighted a greater role for astrocytes in clear-
ing synaptic debris and microglia for somatic debris (83). To assess
astroglial engulfment of synaptic elements, brain samples from
12- to 13-month-old WT and dKI animals were costained with
SV2A and GFAP after 1 month of treatment with vehicle or SAM
mGluR5 SAM (BMS-984923) blocked this AD process, while
preserving physiological glutamate signaling, and had a broad safety
margin in toxicology studies. Thus, SAM-mediated correction of
neuronal expression blocked C1Q synaptic localization and pre-
vented synaptic engulfment to preserve synapses in rodent models.
Synapse loss is a robust biomarker of AD progression that
correlates well with cognitive decline (8–12). Development of a non-
invasive in vivo imaging approach to quantify synaptic density
homogenously expressed isoform of SV2, is found in synapses
used longitudinal PET imaging with [18F]SynVesT-1 to first demon-
strate decreases in hippocampal synaptic density of two separate
mouse models of amyloidogenesis and AD and then to show
pharmacological rescue. These findings were confirmed using
immunohistochemical staining of SV2A. The successful application
of [18F]SynVesT-1 in preclinical evaluation of treatment advances
its utility as a tool for development of therapeutic agents of AD and
other neurodegenerative diseases, and supports its development as
a tool for diagnosis and assessment of AD disease progression.
Pharmacological rescue of synaptic density in aged APP/PS1
and dKI mice revealed that new synapses are being formed in these
animals. It is documented by repeated in vivo two-photon imaging
that aged WT mice (86) and aged APP/PS1 mice (87) continue to

Downloaded from https://www.science.org at Yale University on June 14, 2022

(Fig. 7, F and G). SAM-treated dKI samples exhibited significantly form dendritic spines and stabilize new synapses. Because new
(P  <  0.05) lower SV2A signal contained within GFAP-positive dendritic spines are continually gained and stabilized throughout
astrocytes in CA1, relative to dKI-vehicle samples. Although SAM-­ aging, it is potentially possible to restore overall synaptic density
treated dKI samples did exhibit reductions in astrocytic engulfment by blocking the mechanisms underlying accelerated synaptic loss
of SV2A, the engulfment was still significantly higher than WT without an effect on synaptogenesis per se. On the basis of the acute
samples (Fig. 7G; P < 0.0001). Together, SAM blockade of mGluR5-­ effect of Ao to drive dendritic spine loss and the ability of SAM
dependent and AD-selective synaptic signaling normalizes neuronal treatment to prevent this acute loss (19,  36,  39), we favor the
gene expression and is associated with reductions of both C1Q hypothesis that synaptogenesis is not altered by SAM but rather
synaptic tagging and histologic evidence of synaptic engulfment. that increased density over time is due to a shift in the balance
of gain and loss. The observation that SAM treatment of WT
animals did not result in greater synaptic density is also consistent
DISCUSSION with a specific action to limit AD-related synaptic loss and thereby
In the current study, we showed that synapse loss can be tracked permit a net increase of synapse density over 1 month in the
using longitudinal PET imaging in rodents. Using this method, we disease model.
found that synapse loss was mGluR5-dependent in both a MAPT In addition to synapse loss, microscopic pathological hallmarks
gene KI AD mouse model and a TG amyloidogenic mouse model. of AD in humans include A plaques and neurofibrillary tangles
The mGluR5 intervention was initiated after synapse density had composed of hyperphosphorylated TAU (2,  3). APP/PS1 mice
declined at 12 months of age, and SAM treatment was able to re- recapitulate amyloid phenotypes but do not develop TAU tangles.
verse the deficit in reimaged mice. This disease-modifying benefit To assess SAM-mediated rescue of pathological TAU phenotypes,
persisted for 1 month after washout and was coupled with reduced we previously treated 3×Tg mice (39). These mice express the
pTAU accumulation by dKI mice expressing human TAU. Mecha- human MAPT (P301L) transgene in addition to the APPswe transgene
nistically, single-nuclei transcriptomic profiling revealed normaliza- and a Psen1 transgene with the familial AD (fAD) M146V mutation
tion of neuronal signatures, but little change in glial cells. However, (88). SAM treatment of aged 3×Tg mice reduced soluble p(S199/
this does not imply that glial responses are separable from AD-­ S202)-TAU and insoluble TAU. To extend this finding to a setting
related synapse loss. In contrast, the mGluR5-mediated neuronal without overexpression or FTLD-mutant TAU, we used the homo-
changes eliminated the AD-dependent localization of C1Q to zygous dKI model APP NL-G-F /hMAPT. We confirmed pTAU
synaptic sites and reduced the AD-associated synaptic fragment accumulation and found that pTAU phenotypes were decreased by
engulfment by astrocytes. Our data support the hypothesis that SAM treatment. We have previously shown that SAM treatment
the synapse-depleting neuroglial interaction in these amyloidogenic prevents aberrant activation of proline-rich tyrosine kinase 2 (Pyk2,
and AD models is prevented by mGluR5 blockade–dependent nor- PTK2B) (39), a kinase linked to AD risk and reported to phosphorylate
malization of neuronal gene expression. However, we cannot rule TAU at Y18 (89). In the amyloidogenic and AD models studied
out additional effects of SAM treatment on nonneuronal cells that here, SAM treatment lowered A-triggered TAU phosphorylation,
were not detected by extensive expression profiling or immuno- at least in part by normalization of downstream Pyk2. SAM-­
histology but contributed to synaptic density restoration. Of trans- mediated reduction of pTAU accumulation may contribute to
lational relevance, a subnanomolar potent and orally available restoration of synaptic density.

Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022 11 of 16
SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
Previous studies support sex-dependent differences in AD
incidence. Using APP/PS1 mice, a recent study found that negative
allosteric modulation of mGluR5 selectively improved cognitive
decline in male but not female mice. However, our SV2A PET studies
and extensive histological data found no difference between sexes in
multiple brain regions using the same APP/PS1 background or the
dKI model. One explanation for the difference in sex dependency
between these studies relates to the use of a SAM in the current
study at full receptor occupancy, in contrast to a NAM with atten-
dant toxicity and dose limitation in the previous work. Pharmaco-
kinetic and receptor occupancy were not monitored in the CTEP
study (54). Nonetheless, it is clear that both male and female AD
mice of different strains benefit from SAM treatment target-
ing mGluR5.
The list of genes altered in the AD mouse neurons and corrected
by SAM overlaps strongly with data from human AD expression
profiling. Nineteen of the 46 SAM-corrected ExN DEGs common
to both models are human dorsolateral prefrontal CX ExN DEGs
(90). A number of these genes have also been reported as AD
biomarkers. Aldolase, fructose-bisphosphate A (ALDOA) function
is down-regulated in AD hippocampal tissue (91) and is a CSF
biomarker discriminating between AD and non-AD cognitive im-
pairment (92). Calmodulin (CALM1) is part of an intersecting sub-
network from four consensus modules of genome-wide association
studies (GWAS) for CSF t-TAU/A1–42 ratio in the Alzheimer’s
Disease Neuroimaging Initiative (ADNI) (93). CYSC (CST3) cor-
relates with CSF concentrations of A42 and TAU (94) and is re-
duced in AD CSF (95). Prostaglandin D2 synthase/-trace (PTGDS)
is also reduced in AD CSF (95) and is part of network module with
a positive correlation to AD in frontal CX tissue (96). The ATPase
subunit ATP1B1 is included in a network module with a negative
correlation to AD phenotypes (96). Malate dehydrogenase 1 (MDH1)
is elevated in AD CSF (97) and is a hub in gene networks contribut-
ing to AD (98). Heat shock protein 90 alpha family class B member 1
(HSP90AB1), a molecular chaperone, is a subnetwork node within an
“amyloid neuropathies” network of AD (51). Integral membrane
protein 2B (ITM2B) regulates glutamate release and synaptic func-
tion (99), whereas dominant ITM2B mutations cause familial British
and Danish dementias, diseases with amyloid angiopathy, and neuro-
fibrillary tangles (100). BRI2 (encoded by ITM2B) binds APP and
inhibits A42 aggregation (100). BRI2 is also increased in early-stage
AD HC, and deposits are associated with A plaques (101). CAMK2N1
is a potent and specific inhibitor of CAMKII in the postsynaptic den-
sity. CAMKII is downstream of the PRPC-mGluR5 signaling com-
plex aberrantly activated by Ao (38). Dysregulation of hippocampal
CAMKII has been reported in AD (102).
Despite synapse recovery by SAM treatment, microglial and astro-
cyte gene expression and histologic changes in the amyloidogenic
and AD models were altered by SAM to a minimal extent. Although
there was a relative absence of glial changes, we cannot rule out
direct effects of SAM on glial action as a potential contributing
factor. However, even if SAM directly acts on glial cells, most
AD-related transcriptional changes were observed selectively in
neurons. This is consistent with glial activation being driven by
amyloid accumulation rather than synaptic damage (103). Subsequent
to glial activation in control AD mice, C1Q production tags synapses
for engulfment by both astrocytes and microglia (7, 104). C1Q and
C3 were found to be up-regulated and colocalized to synapses in
AD models, whereas inhibition of classic complement components
Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022
blocked synapse loss. Consistent with these findings, we show here
that preventing localization of C1Q to synapses by SAM treatment
is sufficient to prevent synaptic engulfment. snRNA-seq analysis
demonstrated that SAM treatment had the strongest effect in neu-
rons, with minimal alterations to expression changes in glial cells.
Specifically, neuronal SAM–corrected DEGs are enriched in GO
terms related to synapses. Biochemical and histological analysis
showed that C1Q, IBA1, and GFAP amounts were not altered by
SAM treatment, supporting the transcriptomic data. Despite the
persistence of gliosis and plaque density in SAM-treated samples,
we showed elimination of the synaptic localization of C1Q and a
reduction of synaptic engulfment markers in astrocytes. The
observed reduction of synaptic engulfment by GFAP-positive cells
may be sufficient for synaptic recovery by itself, although it is
also possible that SAM treatment reduces engulfment by other glial
populations not examined here, including microglia and other
GFAP-negative phagocytic cells. Both multiple epidermal growth
factor–like domains 10 (Megf10), a C1Q receptor, and MerTk
contribute to astrocytic clearance of neuronal debris and synap-
tic elimination in developing and adult CNS (77, 105). However,
no expression changes in these receptors were detected in neu-
rons in the current analysis. Among the genes differentially
expressed in both amyloidogenic and AD models and rescued by
SAM in both ExNs and InNs is CD47, a protein shown to inhibit
Downloaded from https://www.science.org at Yale University on June 14, 2022
complement-­dependent synaptic pruning (106). Thus, normal-
ization of the phagocytosis-inhibiting signal CD47  in neurons
by SAM may contribute to synaptic protection. Although CD47 is
normalized by SAM treatment in both models, the direction of dif-
ferential expression in the two models is opposite (up-regulated
in APP/PS1 neurons, down-regulated in dKI neurons), perhaps
due to a later disease stage for APP/PS1 versus dKI. Regardless,
rescuing the altered neuronal expression profile prevented
synaptic tagging, effectively blocking complement-dependent
synaptic loss.
Our study has limitations. Although we used two different
strains, the data are derived from murine models that do not fully
recapitulate human AD. In particular, the TAU phenotypes and cell
death in these models are limited compared to the human condi-
tion. Human AD also has a substantially slower progression and
much longer course than any murine model. The mouse strains are
inbred, and mice were housed in conditions relatively free of
pathogens. Thus, the translation of these studies to human requires
detailed clinical investigation.
Although our snRNA-seq data were supported by histological
evidence, most identified DEGs were not validated. Additional
experiments are necessary to confirm the transcriptional changes
and investigate whether these changes manifest at the protein level.
Such experiments will also shed light on the mechanistic under-
pinnings of synaptic engulfment, including specific C1Q receptors
and the responsible cell types.
Although SAM rescue is mediated by mGluR5, we cannot rule
out contributions of other A cell surface receptors to neurodegen-
eration. This could potentially explain the failure of SAM treatment
to fully rescue the observed increases in pTAU in the dKI model.
Despite this, synaptic density was restored to WT levels in dKI
mice, so these contributions must not be essential.
In conclusion, SAM is a potent and selective inhibitor of the
Ao-induced mGluR5 complex. Toxicological studies showed
safety and tolerability at doses more than 250-fold above the
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SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE
dose required for 50% receptor occupancy in primate brain and for
rescue of pathophysiological TAU, C1Q synaptic localization, synap-
tic engulfment, and synaptic depletion in mouse amyloidogenic and
AD models. This work further reinforces that targeting an Ao-­
induced mGluR5 complex can modify the course of AD and sup-
ports the advancement of SAM intervention into human testing
(ClinicalTrials.gov, NCT04805983). SAM treatment was shown to
have a disease-modifying benefit, because synaptic density is main-
tained even after a month of drug washout. Because gliosis and
plaque density are unchanged by SAM treatment, SAM interven-
tion was still effective even after plaque accumulation. As such,
there is potential for synergy with pharmacological treatments that
suppress gliosis and/or target A or TAU. Future work should
explore the molecular details of C1Q synaptic recruitment, the
mechanisms of synaptic engulfment, and the therapeutic develop-
ment of the SAM compound.
MATERIALS AND METHODS
Study design
The objective of this study was to investigate the ability of an
mGluR5 SAM (BMS-984923) to restore synaptic density and the
underlying molecular mechanisms. For nonclinical pharmacology,
drug metabolism and pharmacokinetics (DMPK), and toxicology
experiments, multiple cell lines and animal models were used (Sup-
plementary Materials). To assess synaptic density, rescue, and its
mechanisms, we treated amyloidogenic AD mice and subjected them
to PET imaging and behavioral assays; after sacrifice, tissue was
used for immunohistochemistry, biochemistry, and snRNA-seq.
Research methods and results are reported in compliance with
the Animal Research: Reporting of In Vivo Experiments (ARRIVE)
guidelines. Sample sizes were chosen on the basis of previous re-
sults to enable adequate statistical power. Mice were random-
ized to treatment groups using the standard = RAND() function
in Microsoft Excel, and groups were matched for age and gender
(fig. S1). Details about sample number for each experiment are
outlined in fig. S1G and in the relevant figure legend. For experi-
mental groups with n < 20, individual data values are provided in
data file S3. All experiments were conducted in a blinded fashion
with respect to genotype and treatment. Outliers were identified in
PRISM by ROUT and removed. For specific experiments, see rele-
vant Materials and Methods section for exclusion criteria, outcome
measures, statistical methods, and experimental procedures. Re-
agents and experimental animals are summarized in data file S4.
Statistics
All results are presented as means ± SEM, unless otherwise
specified. GraphPad Prism 9 software was used for statistical analy-
sis. Data were analyzed using unpaired t tests and one-way,
two-way, or three-way analysis of variance (ANOVA), followed
by post hoc Tukey’s multiple comparisons test, Dunnett’s multiple
comparisons test, Holm-Šídák’s multiple comparisons test, or
Fisher’s least significant difference post hoc pairwise compari-
sons test as specified in the figure legends. Only two-sided tests
were used, and all data analyzed met the assumption for the
specific statistical test that was performed. Probability levels
of P < 0.05 were considered statistically significant. Statistical
tests for gene expression in single-nuclei data were carried out in
Seurat V3.6 (107).
Spurrier et al., Sci. Transl. Med. 14, eabi8593 (2022) 1 June 2022
SUPPLEMENTARY MATERIALS
www.science.org/doi/10.1126/scitranslmed.abi8593
Materials
Figs. S1 to S15
Data files S1 to S4
MDAR Reproducibility Checklist
References (108–120)
View/request a protocol for this paper from Bio-protocol.
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Acknowledgments: We thank T. Saito and T. C. Saido for APPNL-G-F/hMAPT mice. We thank
K. DeLuca and the Yale PET Center staff for their expert technical support. Funding: This
work was supported by research grants from the NIH to Z.C. (R01AG069921), to S.M.S.
(U01AG058608, R01AG034924, R01AG066165, RF1AG070926, and R56AG074015), and to
A.H.B. (F31AG066483, T32NS007224, and T32NS041228), and by the Thome Memorial
Foundation and the Falk Medical Research Trust to S.M.S. Author contributions: J.S. designed
experiments, wrote the first draft of the manuscript, and completed the mouse dosing, tissue
handling, and snRNA-seq. L.N. and L.Z. processed the snRNA-seq data. A.J.S., W.L., M.A.A., and
M.C. completed histological assessments of brain. J.S. and M.C. completed mouse behavior.
X.T.F., T.T., D.H., P.S., S. Lee, and S. Li completed SynVesT-1 PET and mouse FPEB studies. D.H.
completed monkey FPEB PET studies. T.R.S. supervised toxicological studies. A.H.B., H.T., S.H.N.,
and A.P.C. contributed to histology and biochemistry. Y.H.H., R.E.C., and Z.C. supervised the
PET studies, whereas S.M.S. designed the study, wrote the paper, and supervised all aspects.
Competing interests: T.R.S. is an employee, and S.M.S. is founder, equity holder, director, and
paid consultant of Allyx Therapeutics, which seeks to develop mGluR5 compounds for AD and
holds a license for BMS-984923. Y.H.H., Z.C., S. Li, and R.E.C. are inventors on the patent
application (PCT/US2018/018388) submitted by Yale University that covers the production
and use of the radioligand [18F]SynVesT-1 (formerly referred to as [18F]SDM-8). T.R.S. and S.M.S.
are inventors on a planned patent application to be submitted jointly by Yale University and
Allyx Therapeutics that covers the production and use of BMS-984923 for neurodegenerative
disease. S.M.S. also consults for ReNetX Bio and Proteowise on unrelated topics. Data and
materials availability: All data associated with this study are present in the paper or the
Supplementary Materials. The snRNA-seq data are available from NCBI GEO database,
accession number GSE171095.
Submitted 5 April 2021
Resubmitted 6 December 2021
Accepted 25 March 2022
Published 1 June 2022
10.1126/scitranslmed.abi8593
16 of 16
 Reversal of synapse loss in Alzheimer mouse models by targeting mGluR5 to
prevent synaptic tagging by C1Q
Joshua SpurrierLaShae NicholsonXiaotian T. FangAustin J. StonerTakuya ToyonagaDaniel HoldenTimothy R.
SiegertWilliam LairdMary Alice AllnuttMarius ChiasseuA. Harrison BrodyHideyuki TakahashiSarah Helena NiesAzucena
Pérez-CañamásPragalath SadasivamSupum LeeSongye LiLe ZhangYiyun H. HuangRichard E. CarsonZhengxin
CaiStephen M. Strittmatter

Sci. Transl. Med., 14 (647), eabi8593. • DOI: 10.1126/scitranslmed.abi8593

A silent modulator for AD

The loss of synapses observed in patients with Alzheimer’s disease (AD) contributes to the progressive cognitive
impairments. Although microglia activity has been shown to participate in synaptic loss, the mechanisms mediating
the process are not completely elucidated. Now, Spurrier et al. evaluated the role of metabotropic glutamate receptor
5 (mGluR5) on synaptic loss and showed that oral administration of an mGluR5 silent allosteric modulator (SAM)
restored synaptic density and reduced phosphorylated TAU accumulation in mouse models of AD. Mechanistically,
the treatment reversed gene expression changes induced in AD mice and prevented synaptic localization of the

Downloaded from https://www.science.org at Yale University on June 14, 2022

complement protein C1Q. The results suggest that SAMs targeting mGluR5 could be an effective approach for limiting
AD-related synaptic loss.

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