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PNAS
2023 Vol. 120 No. 24 e2301312120
https://doi.org/10.1073/pnas.2301312120
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INAUGURAL ARTICLE
|
Significance
Carbohydrates (or glycans) play
crucial roles in biological systems
ranging from energy storage to
pathogen evasion. However, the
contributions of specific glycans
to brain functions such as
emotion and cognition remain
largely unknown. Here, we show
that 4-
O
-sulfated chondroitin
sulfate (CS) regulates
perineuronal nets (PNNs) and
excitatory–inhibitory synapses in
the mouse CA2 (cornu ammonis
2) hippocampus, a brain region
critical for social memory.
Ablation of CS 4-
O
-sulfation in
adult mice caused malformation
of PNNs, elevated anxiety, and
impaired social memory.
Modulations that reversed the
PNN abnormalities or
replenished 4-
O
-sulfation
restored normal mood and social
cognition. These findings identify
roles for chondroitin
4-
O
-sulfation in higher-order
brain functions and suggest a
potential strategy to address
neurological disorders with social
cognitive dysfunction.
Author contributions: H.H., Y.O., and L.C.H.-W. designed
research; H.H., Y.Z., G.M.M., and G.C.Z. performed
research; H.H., Y.Z., Y.O. and L.C.H.-W. analyzed data;
and H.H., A.M.J., and L.C.H.-W. wrote the paper.
Reviewers: J.S., Case Western Reserve University; and
C.-H.W., The Scripps Research Institute.
The authors declare no competing interest.
Copyright © 2023 the Author(s). Published by PNAS.
This article is distributed under
Creative Commons
Attribution-NonCommercial-NoDerivatives License 4.0
(CC BY-NC-ND)
.
1
To whom correspondence may be addressed. Email:
lhw@caltech.edu.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2301312120/-/DCSupplemental
.
Published June 6, 2023.
CHEMISTRY
NEUROSCIENCE
Chondroitin 4-
O
-sulfation regulates hippocampal perineuronal
nets and social memory
Huiqian Huang
a,b
, Amélie M. Joffrin
a
, Yuan Zhao
c
, Gregory M. Miller
a
, Grace C. Zhang
a
, Yuki Oka
c
, and Linda C. Hsieh-Wilson
a,1
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2022.
Contributed by Linda C. Hsieh-Wilson; received January 24, 2023; accepted April 26, 2023; reviewed by Jerry Silver and Chi-Huey Wong
Glycan alterations are associated with aging, neuropsychiatric, and neurodegenerative dis-
eases, although the contributions of specific glycan structures to emotion and cognitive func
-
tions remain largely unknown. Here, we used a combination of chemistry and neurobiology
to show that 4-
O
-sulfated chondroitin sulfate (CS) polysaccharides are critical regulators of
perineuronal nets (PNNs) and synapse development in the mouse hippocampus, thereby
affecting anxiety and cognitive abilities such as social memory. Brain-specific deletion of CS
4-
O
-sulfation in mice increased PNN densities in the area CA2 (cornu ammonis 2), leading
to imbalanced excitatory-to-inhibitory synaptic ratios, reduced CREB activation, elevated
anxiety, and social memory dysfunction. The impairments in PNN densities, CREB activity,
and social memory were recapitulated by selective ablation of CS 4-
O
-sulfation in the CA2
region during adulthood. Notably, enzymatic pruning of the excess PNNs reduced anxiety
levels and restored social memory, while chemical manipulation of CS 4-
O
-sulfation levels
reversibly modulated PNN densities surrounding hippocampal neurons and the balance of
excitatory and inhibitory synapses. These findings reveal key roles for CS 4-
O
-sulfation in
adult brain plasticity, social memory, and anxiety regulation, and they suggest that targeting
CS 4-
O
-sulfation may represent a strategy to address neuropsychiatric and neurodegener-
ative diseases associated with social cognitive dysfunction.
glycosaminoglycans (GAGs) | glycans | chondroitin sulfate (CS) | perineuronal nets (PNNs) |
social memory
Glycans, like nucleic acids and proteins, are ubiquitous in nature and play crucial roles
in biological processes such as development, host–pathogen interactions, and immune
regulation (1–4). The mammalian central and peripheral nervous systems provide a
rich source of diverse glycans. Large-scale analyses have revealed spatial and temporal
variations in glycan expression across the brain (5) and identified over
4,000
N-glycosylation sites on more than 1,500 glycoproteins (6, 7). Despite being
the most structurally diverse and rapidly evolving class of macromolecules (8), glycans
remain understudied, and their biological functions in the nervous system are insuffi-
ciently understood.
In the brain, a complex meshwork of interwoven glycans and proteins in the extra-
cellular matrix (ECM) provides structural support and mediates important neural func
-
tions (9–11). Lattice-like ECM structures known as perineuronal nets (PNNs) have
been proposed to act as extracellular scaffolds for ligands and to stabilize synapses,
thereby modulating brain plasticity and physiological processes (10, 11). Emerging
evidence suggests that PNNs contribute not only to critical period plasticity during
development but also to learning and memory processing, psychiatric diseases, drug
addiction, and neurodegeneration in adulthood (12–14). PNNs predominantly sur-
round parvalbumin-expressing (PV
+
), fast-spiking inhibitory interneurons in cortical
areas (15, 16). They also enwrap excitatory pyramidal neurons in brain regions important
for emotional learning and memory, such as the amygdala, entorhinal cortex, and area
cornu ammonis 2 (CA2) (17, 18).
The CA2 subregion of the hippocampus has unique molecular, synaptic, and morpho-
logical characteristics (19) and is essential for social recognition memory (20, 21).
Consistent with this function, alterations in the cellular structure and circuitry in the area
CA2 have been observed in neuropsychiatric disorders associated with cognitive and social
dysfunction (22, 23). Although recent studies suggest that PNNs in the CA2 can con-
tribute to both PV
+
interneuron and excitatory pyramidal neuron plasticity (24, 25), the
molecules and mechanisms that regulate PNNs and CA2-dependent functions such as
social memory remain unclear.
PNNs are highly enriched in chondroitin sulfate proteoglycans (CSPGs), a series of
core proteins decorated with chondroitin sulfate (CS) polysaccharides (26). The repeating
d-glucuronic acid (GlcA) and
N
-acetyl-d-galactosamine (GalNAc) disaccharide units of
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CS polysaccharide chains display a variety of sulfation motifs
(Fig. 1
A
) that dynamically change throughout development into
adulthood (27). Notably, increased expression of the 4-
O
-sulfated
motif CS-A coincides with PNN maturation and the end of crit-
ical period plasticity (16, 28). By adulthood, the CS-A motif rep-
resents nearly 90% of the total CS in the brain. The 4-
O
-sulfated
CS-A and CS-E motifs have also been reported to play important
roles in axon regeneration after central nervous system (CNS)
injury (29–31). These motifs are up-regulated in the glial scar
upon injury and limit axon regeneration by interacting with pro-
tein receptors such as the tyrosine phosphatase receptor
σ
(PTP
σ
)
and Nogo receptor (29, 32–34). Loss of 4-
O
-sulfated CS motifs
via knockdown of the chondroitin-4-
O
-sulfotransferase
Chst11
in
zebrafish enhanced regeneration after spinal cord injury (31).
Based on these and other observations, chondroitin 4-
O
-sulfation
has traditionally been viewed as a “molecular brake” that inhibits
neuroplasticity (35, 36). However, its functions in PNNs and in
the uninjured adult brain have not been directly investigated.
Studies aimed at understanding PNNs have employed either chon
-
droitinase ABC (ChABC) to enzymatically digest CS polysaccharides
(37–39) or transgenic mice lacking PNN proteins such as tenascin-R
and link protein (HAPLN1) (40, 41). While these approaches have
revealed important insights, they also drastically disrupt PNNs and
render them indistinguishable from the diffuse ECM. Modulation
of the sulfation patterns on CSPGs provides a less perturbative, com
-
plementary approach to manipulate and study PNNs. For example,
overexpression of chondroitin-6-
O
-sulfotransferase-1 (C6ST-1) in
transgenic mice modulated 6-
O
-sulfation and PNN formation in the
developing visual cortex (VC), leading to persistent ocular dominance
plasticity (16). However, the role of CS sulfation in PNNs has been
examined primarily in this context of 6-
O
-sulfotransferase
overexpression.
In this study, we investigated the impact of CS 4-
O
-sulfation, the
dominant form of sulfation and the most abundant glycosamino-
glycan structure in the adult mammalian brain, on PNNs, plasticity,
and higher-order brain functions such as mood and cognition. We
found that brain-specific deletion of the chondroitin
4-
O
-sulfotransferase gene
Chst11
in mice significantly perturbed
PNN levels surrounding excitatory CA2 pyramidal neurons. The
resulting increase in PNNs led to reduced CREB activation, an
imbalance of excitatory and inhibitory synapses, as well as anxiety
and social memory dysfunction—phenotypes that were rescued by
treatment with ChABC or 4-
O
-sulfated CS polysaccharides. In
agreement with these findings, a chemical inhibitor developed by
our lab to reduce CS 4-
O
-sulfation levels recapitulated the malfor-
mation of PNNs and synaptic defects in hippocampal neurons.
Moreover, viral-mediated CA2 region-specific deletion of
Chst11
in
adult mice also increased PNN densities, inhibited CREB activity,
and impaired social memory. Together, these data reveal important
roles for CS 4-
O
-sulfation in adult brain plasticity, social memory,
and anxiety regulation, and they suggest CS polysaccharides as targets
for the study and potential treatment of neurological diseases char-
acterized by mood disorders and social dysfunction.
Results
Chst11cKO Mice Lacking 4-
O
-Sulfation Show Increased WFA
+
PNN
Densities in the CA2 Hippocampus.
The sulfotransferase Chst11
selectively transfers a sulfate group from 3
-phosphoadenosine
5
-phosphosulfate to the 4
O
-position of GalNAc residues in CS
polysaccharides (42). Genetic disruption of
Chst11
in mice led
to severe chondrodysplasia and neonatal lethality, demonstrating
essential roles for 4-
O
-sulfation in embryonic development (43,
44). To study the roles of CS 4-
O
-sulfation in the adult brain, we
generated a brain-specific
Chst11
conditional knockout (cKO)
mouse line by crossing
Chst11
-floxed mice with
nestin
-
Cre
transgenic
mice (45). The
nestin
-
Cre
transgene restricts
Chst11
deletion to neural
precursor cells starting at embryonic day 10.5 (E10.5), circumventing
complications associated with constitutive
Chst11
ablation during
prenatal development (43). To validate the gene deletion efficiency,
we analyzed the sulfation patterns of CS in the cortex of
Chst11
lx/−
;
nestin-Cre
+
(Chst11cKO) mice and
Chst11
lx/−
; nestin-Cre
(Ctrl) mice
as a control. CS chains were isolated from Chst11cKO and Ctrl
mouse cortices at different ages (P0, P7, P14, P28, and P60), digested
with ChABC, and the resulting disaccharides were quantified by
high-performance liquid chromatography. No 4-
O
-sulfated CS-A
and CS-E disaccharide units were detected in Chst11cKO brains,
indicating disruption of the 4-
O
-sulfation pathway (Fig. 1
A
and
B
). Loss of chondroitin 4-
O
-sulfation was accompanied by an
increase in unsulfated CS levels and a decrease in overall CS levels
in the brain (
SI Appendix
, Fig. S1
A
), consistent with reports that
4-
O
-sulfation facilitates CS chain elongation (46). In contrast, no
loss of 6-
O
-sulfation was observed: CS-D levels remained similar,
and CS-C levels increased slightly in Chst11cKO mice compared
to Ctrl mice after P14, presumably due to compensatory effects
(
SI Appendix
, Fig. S1
A
). Disruption of the 4-
O
-sulfation pathway in
Chst11cKO mice was further confirmed by immunohistochemical
analysis. A marked reduction in CS-E immunostaining was observed
in the cortex and hippocampus of Chst11cKO mice relative to
Ctrl littermates (
SI Appendix
, Fig. S1
B
and
C
). Collectively, these
results indicate efficient deletion of the 4-
O
-sulfation pathway in
Chst11cKO mice.
We next examined the PNN levels in the VC and hippocampus of
adult Chst11cKO and Ctrl mice using the well-established marker,
Wisteria floribunda
agglutinin (WFA) (16, 18, 37, 39). Consistent with
previous observations, WFA
+
PNNs exhibited region-specific expres-
sion patterns (47). In the VC, an increase in PNN-enwrapped (WFA
+
)
neurons was observed in adult Chst11cKO mice compared to Ctrl
mice (36% ± 11%), and this increase occurred primarily in PNNs
surrounding the PV
+
population (
SI Appendix
, Fig. S2
A
C
). No
change was observed in the total number of PV
+
neurons or its per-
centage within the WFA
+
population (
SI Appendix
, Fig.
S2
D
and
E
).
Notably, the greatest change in WFA
+
PNN-enwrapped neurons
was observed in the area CA2 of the hippocampus. Both Chst11cKO
and Ctrl adult mice showed high PNN levels in the CA2 region,
whereas the CA1 and CA3 areas had fewer WFA
+
PNNs (Fig.
1
C
).
A striking increase in the number of WFA
+
PNNs was observed in
the CA2 region (109% ± 15%) and, to a lesser extent, in the CA3
region (55% ± 13%) of Chst11cKO compared to Ctrl mice (Fig.
1
C
).
Given the substantial increase in WFA
+
PNNs detected in the CA2
and the limited understanding of CS function in this region, we
focused our studies hereafter on the CA2 hippocampus.
As PNNs condense around PV
+
inhibitory neurons in many
brain regions (16, 48), we investigated whether the WFA
+
PNNs
in the CA2 surround PV
+
neurons. Adult hippocampal sections
were costained for PV and the CA2 marker Purkinje cell protein
4 (PCP4) (49). The PCP4
+
neurons in the CA2 region of both
Chst11cKO and Ctrl mice contained only a small percentage of
PV
+
neurons (8% ± 2%; Fig.
1
D
,
Upper Right
). The majority of
the PCP4
+
neurons in Chst11cKO mice were enwrapped by WFA
+
PNNs (95% ± 2%; Fig.
1
D
,
Lower Right
), whereas fewer PCP4
+
neurons in Ctrl mice were surrounded by WFA
+
PNNs (33% ±
8%). These findings are consistent with previous reports that
PNN-enwrapped excitatory neurons are prevalent in the CA2
hippocampus (47, 49) and indicate that CS 4-
O
-sulfation regu-
lates the density of PNNs surrounding excitatory CA2 neurons.
Next, we examined potential mechanisms by which loss of
4-
O
-sulfation could alter PNNs in the adult hippocampus.
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