Child abuse associates with increased recruitment of perineuronal nets in the ventromedial prefrontal cortex: evidence for an implication of oligodendrocyte progenitor cells

Abstract

Child abuse (CA) is a strong predictor of psychopathologies and suicide, and can lastingly alter normal trajectories of brain development, particularly in areas closely linked to emotional responses such as the prefrontal cortex (PFC). Yet, the cellular underpinnings of these enduring effects are unclear. Childhood and adolescence are marked by the protracted formation of perineuronal nets (PNNs), which are essential in orchestrating the closure of developmental windows of cortical plasticity by regulating the functional integration of parvalbumin interneurons (PV) into neuronal circuits. Using well-characterized post-mortem brain samples, we explored the hypothesis that CA has lasting effects on the development of PNNs in the ventromedial PFC. We found that a history of CA was specifically associated with increased recruitment and maturation of PNNs. Through single-nucleus sequencing and fluorescent in-situ hybridization, we show that the expression of canonical components of PNNs is highly enriched in oligodendrocyte progenitor cells (OPCs), and that they are upregulated in CA victims. These findings suggest that early-life adversity may lead to persistent patterns of maladaptive behaviours by reducing the neuroplasticity of cortical circuits through the enhancement of developmental OPC-mediated PNN formation.

Full text

Child abuse associates with increased recruitment of perineuronal nets in the ventromedial
prefrontal cortex: evidence for an implication of oligodendrocyte progenitor cells
Arnaud Tanti1,2, Claudia Belliveau1,3, Corina Nagy1, Malosree Maitra1,3, Fanny Denux1, Kelly Perlman1,3,
Frank Chen1, Refilwe Mpai1,3, Candice Canonne1, Maria Antonietta Davoli1, Gustavo Turecki1,4 and
Naguib Mechawar1,4
Affiliations
1 McGill Group for Suicide Studies, Douglas Mental Health University Institute, Verdun, QC, Canada.
2 Current address: UMR 1253, iBrain, Université de Tours, Inserm, Tours, France.
3 Integrated Program in Neuroscience, McGill University, Montreal, QC, Canada.
4 Department of Psychiatry, McGill University, Montreal, QC, Canada.
Corresponding author
Naguib Mechawar, PhD
McGill Group for Suicide Studies, Douglas Mental Health University Institute
Department of Psychiatry, McGill University
naguib.mechawar@mcgill.ca
Content
Main Text – Abstract, Introduction, Results, figure legends and discussion (1855 words)
Methods (1311 words)
2 figures, 1 table
15 references
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Abstract
Child abuse (CA) is a strong predictor of psychopathologies and suicide, and can lastingly alter normal
trajectories of brain development, particularly in areas closely linked to emotional responses such as the
prefrontal cortex (PFC). Yet, the cellular underpinnings of these enduring effects are unclear. Childhood
and adolescence are marked by the protracted formation of perineuronal nets (PNNs), which are essential
in orchestrating the closure of developmental windows of cortical plasticity by regulating the functional
integration of parvalbumin interneurons (PV) into neuronal circuits. Using well-characterized post-
mortem brain samples, we explored the hypothesis that CA has lasting effects on the development of
PNNs in the ventromedial PFC. We found that a history of CA was specifically associated with increased
recruitment and maturation of PNNs. Through single-nucleus sequencing and fluorescent in-situ
hybridization, we show that the expression of canonical components of PNNs is highly enriched in
oligodendrocyte progenitor cells (OPCs), and that they are upregulated in CA victims.
These findings suggest that early-life adversity may lead to persistent patterns of maladaptive behaviours
by reducing the neuroplasticity of cortical circuits through the enhancement of developmental OPC-
mediated PNN formation.
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Introduction
Child abuse (CA) has enduring effects on psychological development. Severe adversity during sensitive
periods, during which personality traits, attachment patterns, cognitive functions and emotional responses
are shaped by environmental experiences, is thought to have a profound effect on the structural and
functional organization of the brain (1).
At the cellular level, childhood and adolescence are marked by the establishment and maturation of neural
circuits. This protracted period is characterized by windows of heightened plasticity that precede the
development of functional inhibitory connections and the balance of excitatory-inhibitory
neurotransmission (2). A major mechanism behind the closing of these windows in neocortex is the
recruitment of perineuronal nets (PNNs), a condensed form of extracellular matrix forming most notably
around parvalbumin-expressing (PV+) interneurons. PNNs are thought to gradually decrease heightened
plasticity by stabilizing the integration and function of PV+ cells into cortical networks and hindering the
remodelling of these networks (3, 4). This PNN-induced stabilization of PV+ interneuron connectivity has
been notably linked to the persistence of long-term associations, including fear memories (5–7).
Although little is known regarding the cellular effects of CA, evidence in rodents suggests that early-life
stress associates with precocious functional maturation of PV+ neurons and the early emergence of adult-
like characteristics of fear and extinction learning (8), in addition to discrete changes in the
immunoreactivity of inhibitory neuron markers and PNNs (9). Taken together, these observations suggest
that CA may alter the formation of PNNs.
We addressed this question using well-characterized post-mortem samples (Douglas-Bell Canada Brain
Bank) from adult depressed suicides who died during an episode of major depression with (DS-CA) or
without (DS) a history of severe child abuse and from matched psychiatrically healthy individuals
(CTRL). Standardized psychological autopsies were conducted to provide comprehensive post-mortem
diagnostic and retrieve various dimensions of childhood experience, including history and severity of CA.
We focused on the ventromedial prefrontal cortex (vmPFC), a brain area closely linked to emotional
learning and which is structurally and functionally altered in individuals with a history of CA (1).
Results and discussion
PNNs densities, visualized by Wisteria Floribunda Lectin (WFL) labeling and NeuN immunostaining
(Fig.1A) were markedly higher in the vmPFC of individuals with a history of CA (Fig.1B). To
understand whether these changes were directly linked to increased PNNs recruitment rather than changes
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in PV+ cells numbers, we compared ratios of PV+ cells surrounded by PNNs. PV antigenicity is
particularly susceptible to freezing, and lost altogether in samples snap frozen prior to fixation. We
therefore developed an approach to combine fluorescence in-situ hybridization (FISH) and
immunofluorescence to visualize PVALB expressing cells and PNNs in frozen samples (Fig.1C). ~65%
of PVALB+ cells were surrounded by PNNs (Fig.1D), in line with previous observations. Importantly,
samples from DS-CA individuals displayed a robust increase in the percentage of PVALB+ cells
surrounded by PNNs compared to DS and CTRL samples (Fig.1D).
Figure 1. (A) Representative images of PNNs labeled with WFL and their distribution throughout the cortical layers of the
human vmPFC. Scale bars = 100 and 20 µm (high magnification panel) (B) Depressed suicides with a history of child abuse (DS-
CA, N = 11) have significantly higher densities of PNNs compared to controls (CTRL, N = 10) and depressed suicides without
history of child abuse (DS, N= 14) (layer x group: F (10, 115) = 2.07, P< 0.05, followed by Tukey’s multiple comparisons test).
(C) Representative images of in situ hybridization for PVALB (green) followed by WFL labeling (red). Nuclei were stained with
DAPI (cyan). Yellow arrowhead points to a PVALB+/PNN- cell. Scale bars = 25µm. (D) DS-CA (N=9) subjects have higher
ratios of PVALB+ cells surrounded by PNNs compared to CTRLs (N =8) and DS subjects (N=4) (Kruskal-Wallis ANOVA
H(2,21) = 9.45, P<0.01, followed by Dunn’s test for two by two comparisons). (E) Representative images of a low (top) and high
(bottom) intensity PNN in the vmPFC. (F) PNNs from DS-CA subjects (N=475; 6) showed higher average WFL intensity
compared to CTRLs (N= 387; 6) or DS (N=479; 5) (One-way ANOVA: F(2, 1338) = 80.56, P<0.001, followed by Tukey’s
multiple comparisons test) (G) PNNs from DS-CA subjects (N=432; 6) showed higher complexity (area covered by PNNs)
compared to CTRLs (N= 363; 6) or DS (N=467; 5) (One-way ANOVA: F (2, 1259) = 11.06, P<0.001, followed by Tukey’s
multiple comparisons test). ***:P<0.001, **: P<0.01, *:P<0.05, #:P<0.1.
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To investigate if CA associates with maturational changes of PNNs, we compared the intensity of WFL
staining between groups (Fig.1E) as an indication of their maturity (10), as well as the area covered by
individual PNNs as an indicator of their morphological complexity. CA was both associated with higher
intensity of WFL staining per PNN (Fig.1F) and cells more extensively covered by PNNs (Fig.1G),
suggesting overall that CA may precipitate both the maturation and the recruitment of PNNs around PV+
cells.
We then sought to explore the molecular underpinnings of this phenomenon, and reasoned that increased
recruitment of PNNs associated with CA should translate or be induced by changes in the molecular
programs controlling PNN assembly. Our understanding of these transcriptional programs is particularly
scarce, hindered by the fact that several known molecules participating in PNN recruitment are released
non-locally and by different cell types, implying a complex cellular crosstalk orchestrating PNN
assembly. To gain insight into how, in humans, different cell types contribute to the synthesis of
canonical components of PNNs, we explored a single-nucleus sequencing dataset previously generated by
our group in the human PFC (11), and screened their expression across 8 major cell types. The main
canonical components of PNNs, namely aggrecan, neurocan, versican, phosphacan, brevican, and
tenascin-R, were highly enriched in oligodendrocyte progenitor cells (OPCs) (Fig.2A). This was further
validated using FISH (Fig.2B and 2C) for versican (VCAN) and phosphacan (PTPRZ1), as they showed
the strongest expression in OPCs and are two major signature genes in late OPCs (12). We found that
virtually all PDGFRA+ OPCs express high levels of these components, while cells expressing these genes
are almost all PDGFRA+ OPCs (Fig.2D and 2E). This strongly suggests that OPCs are likely potent
regulators of PNN formation, as they are also ontogenetically-related to PV+ cells (13) and have been
shown to form extensive synaptic networks with them (14).
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Figure 2. (A) Average gene expression of canonical components of PNNs according to cell type, derived from single-nucleus
RNA sequencing of 34 human prefrontal cortex samples (11). Expression was calculated by weighting normalized transcript
counts of each cluster by the size (number of nuclei) of the cluster. Weighted average expression values are displayed in a
heatmap, with the expression values as z-scores, and darker colors indicating higher expression. OPCs consistently express
higher levels of most of these components compared to other cell types. (B) Representative images of in situ hybridization
validation of VCAN (Versican, yellow) expression in OPCs (PDGFRA+ cells, white). Note the VCAN expressing OPC directly
juxtaposed to a PVALB+ (magenta) cell. Nuclei were counterstained with DAPI (blue). Scale bar = 5 µm. (C) Representative
images of in situ hybridization validation of PTPRZ1 (Phosphacan, yellow) expression in OPCs (PDGFRA+ cells, white). Nuclei
were counterstained with DAPI (blue). Scale bar = 5 µm. (D-E) Both VCAN (D) and PTPRZ1 (E) expression is highly enriched
in OPCs, with 97.8 percent of VCAN+ cells (N=225) co-expressing PDGFRA (D), and 91.8 percent of PTPRZ1+ cells (N= 281)
co-expressing PDGFRA (E). (F) A negative correlation was found between average distance of OPCs from closest PVALB+ cell
and PNNs density (R2 = 0.36, P<0.05) in the same subjects, suggesting that OPCs proximity with PVALB+ cells could reflect
changes in PNN density. (G) Proximity of OPCs with PVALB+ cells was decreased in DS-CA subjects (N=90 OPCs, 5 subjects)
compared to CTRLs (N= 106 OPCs, 6 subjects) and DS (N= 73 OPCs, 4 subjects) (One-way ANOVA: F (2, 266) = 7.963,
P<0.001, followed by Tukey’s multiple comparison test). (H-I) Both PTPRZ1 (H) and TNR (I) average expression in OPCs
modestly correlated with PNNs densities (R2 = 0.35, P<0.05 and R2 = 0.28, P<0.05 respectively). (J) The average expression of
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VCAN in OPCs was significantly higher in DS-CA subjects (N=139 cells, 7 subjects) compared to CTRLs (N= 160 cells, 8
subjects) and DS (N= 119 cells, 6 subjects) (One-way ANOVA F (2, 415) = 17.25, P<0.001, followed by Tukey’s multiple
comparisons test). (K) The average expression of PTPRZ1 in OPCs was significantly higher in DS-CA subjects (N=63 cells, 4
subjects) compared to CTRLs (N= 117 cells, 6 subjects) and DS (N= 81 cells, 5 subjects) (One-way ANOVA F (2, 258) = 31.65,
P<0.001, followed by Tukey’s multiple comparisons test). (L) The average expression of TNR in OPCs was significantly higher
in DS-CA subjects (N=200 cells, 8 subjects) compared to CTRLs (N= 207 cells, 7 subjects) and DS (N= 160 cells, 5 subjects)
(One-way ANOVA, F (2, 564) = 18.69, P<0.001, followed by Tukey’s multiple comparisons test). ***:P<0.001), *:P<0.05.
OPCs were on occasions found to be directly juxtaposed to PVALB+ cells (Fig.2B), as previously
reported in rodents (15). Interestingly, OPC proximity to PVALB+ cells modestly correlated with PNN
density (Fig.2F), and was increased in individuals with a history of CA (Fig.2G), further suggesting an
interplay between these two cell types. In support of a possible involvement of OPCs in mediating CA-
related changes in PNNs, the expression of both PTPRZ1 and TNR in OPCs correlated with PNNs
densities (Fig.2H and 2I) and were upregulated in OPCs of CA victims (Fig.2J-L).
Overall, our results suggest that a history of CA associates with increased recruitment and maturation of
PNNs, as well as an upregulation of their canonical components by OPCs, a cell type that likely plays a
key role in the cellular crosstalk that orchestrates PNN formation. Whether changes in OPCs are causal in
the increased recruitment of PNNs following CA or an indirect response following changes in PNN
dynamics remains to be explored in preclinical models. These findings raise the possibility that aversive
memories and maladaptive patterns of emotional processing associated with early-life adversity could be
stabilized more durably in the brain of CA victims, thus favoring the emergence of psychopathologies.
Acknowledgments
The present study used the services of the Douglas-Bell Canada Brain Bank and of the Molecular and
Cellular Microscopy Platform (MCMP) at the Douglas Hospital Research Centre. The authors are grateful
to Maâmar Bouchouka, Josée Prud’homme, Dominique Mirault, and Melina Jaramillo Garcia for their
kind assistance.
Author Contributions
AT and NM conceived the study. GT participated in the acquisition and clinical characterization of the
brain samples. AT, CB, FD, MAD, CC, RM contributed to immunohistological experiments. AT, CN,
MM and KP generated and analysed the snSeq dataset. AT and FC performed the in situ hybridization
experiments. AT and NM prepared the manuscript and all authors contributed to and approved its final
version.
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Competing Interest Statement
The authors have no financial interest or conflict of interest to declare.
Funding
This work was funded by a CIHR Project grant to NM. AT was supported by fellowships from the FRQS
and Toronto Dominion, and an American Foundation for Suicide Prevention (AFSP) Young Investigator
Innovation Grant (YIG-0-146-17). The Molecular and Cellular Microscopy Platform and the Douglas-
Bell Canada Brain Bank (DBCBB) are partly funded by a Healthy Brains for Healthy Lives (CFREF)
Platform Grant to GT and NM. The DBCBB is also funded by the Réseau Québécois sur le suicide, le
troubles de l’humeur et les troubles associés (FRQS).
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Material and Methods
Human post-mortem brain samples. Brain samples were obtained from the Douglas-Bell Canada Brain
Bank (Montreal, Canada). Phenotypic information was retrieved through standardized psychological
autopsies, in collaboration with the Quebec Coroner’s Office and with informed consent from next of kin.
Presence of any or suspected neurological/neurodegenerative disorder signalled in clinical files
constituted an exclusion criterion. Cases and controls are defined with the support of medical charts and
Coroner records. Proxy-based interviews with one or more informants best acquainted with the deceased
are supplemented with information from archival material obtained from hospitals, Coroner’s office and
social services. Clinical vignettes are then produced and assessed by a panel of clinicians to generate
DSM-IV diagnostic criteria, providing sociodemographic characteristics, social developmental history,
DSM-IV axis I diagnostic information and behavioural traits; information that is obtained through
different adapted questionnaires. Toxicological assessments and medication prescription are also
obtained. Presence of severe child abuse was based on adapted Childhood Experience of Care and Abuse
(CECA) interviews assessing various dimensions of childhood experience, including abuse (Bifulco et al.,
1994).
Table 1. Subjects characteristics
CTRL DS-CA DS
N 11 12 16
Axis 1 diagnosis 0 MDD (11); DD-NOS (1) MDD (14); DD-NOS (2)
Age (years) 43.18 ± 7.11 37.75 ± 3.10 46.63 ± 3.48
Sex (M/F) 9/2 9/3 14/2
PMI (hours) 35.95 ± 7.15 40.92 ± 6.68 45.95 ± 8.06
Tissue pH 6.4 ± 0.09 6.56 ± 0.08 6.52 ± 0.07
Substance dependence 0 5 6
Medication 0
SSRI (2); Benzodiazepines (2);
Antipsychotics (1); Antimanic
(1)
SSRI (4); SNRI (1); TCA
antidepresants (1);
Benzodiazepines (3);
Antipsychotics (2); Antimanic
(1)
Data represent mean ± s.e.m. PMI: Post-mortem interval; MDD: Major Depressive Disorder; DD-NOS: Depressive Disorder Not
Otherwise Specified. SSRI: Selective Serotonin Reuptake Inhibitor; SNRI: Selective Norepinephrine Reuptake Inhibitor; TCA:
tricyclic antidepressant.
Tissue dissections
Dissections were performed by expert brain bank staff on fresh-frozen 0.5 cm thick coronal sections with
the guidance of a human brain atlas. Ventromedial prefrontal cortex samples were dissected in sections
equivalent to plate 3 (approximately − 48 mm from the center of the anterior commissure) of this atlas.
Samples were either kept frozen or fixed overnight in 10% formalin until processed for in situ
hybridization or immunohistochemistry, respectively.
Immunohistochemistry
Frozen tissue blocks were fixed in 10% neutral buffered formalin overnight at 4 °C, rinsed in PBS and
kept in 20% sucrose/PBS solution until serially sectioned at 40 μm on a cryostat. Free-floating sections
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were rinsed in phosphate-buffered saline (PBS) and then incubated overnight at 4ºC under constant
agitation with a mouse anti-NeuN antibody (Millipore, 1:500, MAB377) and biotinylated Wisteria
Floribunda Lectin (WFL, Vector Laboratories, B-1355; 1:2500) diluted in a blocking solution of
PBS/0.2% Triton-X/2% normal donkey serum. Sections were then rinsed and incubated for 2h at room
temperature with Cy3-conjugated Streptavidin (Jackson ImmunoResearch, 016-160-084; 1:500) for the
detection of PNNs and Alexa-488 conjugated anti-Mouse (Jackson ImmunoResearch, 1:500) for NeuN,
diluted in the same blocking solution as the primary incubation. Following the secondary antibody
incubation, sections were rinsed, endogenous autofluorescence from lipofuscin and cellular debris was
quenched with Trueblack (Biotium), and sections were mounted on Superfrost charged slides and
coverslipped with Vectashield mounting medium (Vector Laboratories, H-1800).
Whole vmPFC sections were scanned on a Zeiss Axio Imager M2 microscope equipped with a motorized
stage and Axiocam MRm camera at x20. The ImageJ software (NIH) Cell Counter plugin was used to
manually count PNNs. An average of 4 sections per subject was used. Cortical layers were delineated
based on NeuN+ cells distribution and morphology, and the number of PNNs as well as the area of each
layer were measured, allowing to generate PNNs density values (n/mm²).
Fluorescent in situ hybridization
Frozen BA9 blocks were cut serially with a cryostat and 10µm-thick sections collected on Superfrost
charged slides. In situ hybridization was performed using Advanced Cell Diagnostics RNAscope® probes
and reagents following the manufacturer instructions. Sections were first fixed in cold 10% neutral
buffered formalin for 15 minutes, dehydrated by increasing gradient of ethanol bathes and air dried for 5
minutes. Endogenous peroxidase activity was quenched with hydrogen peroxide for 10 minutes followed
by protease digestion for 30 min at room temperature. The following probes were then hybridized for 2
hours at 40C in a humidity-controlled oven: Hs-PVALB (cat. no. 422181), Hs-VCAN (cat. no. 430071-
C2), Hs-PDGFRA (cat. no. 604481-C3), Hs-TNR (cat. no. 525811), Hs-PTPRZ1 (cat. no. 584781-C2).
Amplifiers were added using the proprietary AMP reagents, and the signal visualized through probe-
specific HRP-based detection by tyramide signal amplification with Opal dyes (Opal 520, Opal 570 or
Opal 690; Perkin Elmer) diluted 1:700. Slides were then coverslipped with Vectashield mounting medium
with DAPI for nuclear staining (Vector Laboratories) and kept at 4C until imaging. For
immunohistochemical staining of PNNs following PVALB in situ hybridization, slides were rinsed in
PBS, incubated overnight at 4C with biotinylated WFL followed by Cy3-conjugated Streptavidin for 2
hours prior to coverslipping.
Imaging and analysis of in situ RNA expression in OPCs
Image acquisitions was performed on a FV1200 laser scanning confocal microscope (FV1200) equipped
with a motorized stage. For each experiment and subject, 6 to 10 stack images were taken to capture at
least 20 OPCs (PDGFRA+) per subject. Images were taken using a x60 objective (NA = 1.42) with a XY
pixel width of ~0.25µm and Z spacing of 0.5µm. Laser power and detection parameters were kept
consistent between subjects for each set of experiment. Since TSA amplification with Opal dyes yields a
high signal to noise ratio, parameters were optimized so that autofluorescence from lipofuscin and cellular
debris was filtered out. OPCs were defined by bright clustered puncta-like PDGFRA signal present
within the nucleus of the cells. Using ImageJ, stacks were first converted to Z-projections, and for each
image the nuclei of OPCs were manually contoured based on DAPI expression. Expression of VCAN,
TNR or PTPRZ1 in OPCs was quantified using the area fraction, whereby for each probe the signal was
first manually thresholded and the fraction of the contoured nucleus area covered by signal measured for
each OPC. Area fraction was the preferred measure to reflect RNA expression as punctate labeling
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generated by FISH often aggregates into clusters that cannot readily be dissociated into single dots or
molecules.
Ratios of PVALB+/PNN+ cells
Proportions of PVALB+ cells enwrapped by PNNs were determined in a single section. An average of 55
PVALB+ cells per subject were imaged under a x20 objective through layers 4-5 of the vmPFC.
Intensity and area measurements
For each subject, ~15 z-stacks (0.26μm Z-spacing) spanning all layers of the vmPFC were acquired at 40x
magnification on an Olympus FV1200 laser scanning confocal microscope. PNNs were traced manually
with ImageJ using maximum intensity projections generated from each stack. For each PNN, the mean
pixel value of adjacent background was subtracted to the mean pixel value of the contoured soma of the
PNN, yielding the mean corrected fluorescence intensity. To infer on their morphological complexity, we
measured the area covered by each contoured PNN, including soma and ramifications extending over
proximal dendrites.
Cell-type specific expression of PNN canonical components using single-nucleus sequencing
Cell-type specific expression of canonical components of PNNs was explored using a snRNA-seq dataset
from the human prefrontal cortex previously generated by our group (11), for which methodology is
extensively described in this published resource. Average expression for each PNN component in each
cell type was calculated by weighting the expression values (normalized transcript counts) of each cluster
by the size (number of nuclei) of the cluster. Weighted average expression values are displayed in a
heatmap, scaled by row (i.e. gene). The color bar therefore represents the expression values as z-scores,
with darker colors indicating higher expression.
Statistical analyses
Analyses were performed on Statistica version 12 (StatSoft) and Prism version 6 (GraphPad Software).
Distribution and homogeneity of variances were assessed with Shapiro–Wilk and Levene’s tests,
respectively. PNNs densities were analyzed using a mixed-effects model, using layer and group as fixed
factors, followed by Tukey’s HSD test for corrected post hoc comparisons. For all other variables (WFL
intensity, WFL area per PNN, PNN+/PVALB ratios, RNA expression in OPCs and distance of OPCs
from PVALB+ cells) group effects were detected using one-way ANOVAs or Kruskal-Wallis test
followed by Tukey’s HSD or Dunn’s test respectively. Linear regressions were used to address the
relationship between PNNs densities, distance of OPCs from PVALB+ cells, and RNA expression of
canonical components of PNNs. Significance threshold was set at 0.05.
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