Proteoglycan abnormalities in olfactory epithelium tissue from subjects
diagnosed with schizophrenia
Harry Pantazopoulosa,b, Anne Boyer-Boiteaub, Eric H. Holbrookc,d, Woochan Jangd, Chang-Gyu Hahne,
Steven E. Arnolde, Sabina Berrettaa,b,f,⁎
aDepartment of Psychiatry, Harvard Medical School, 25 Shattuck St., Boston, MA 02115, USA
bTranslational Neuroscience Laboratory, McLean Hospital, 115 Mill St., Belmont, MA 02478, USA
cDept. of Otology and Laryngology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA
dDepartment of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 2111, USA
eDepartment of Psychiatry, University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104, USA
fProgram in Neuroscience, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
a b s t r a c ta r t i c l ei n f o
Received 1 July 2013
Received in revised form 4 August 2013
Accepted 13 August 2013
Available online 10 September 2013
Chondroitin sulfate proteoglycans
markedly abnormal expression of chondroitin sulfate proteoglycans (CSPGs), key components of the extracellular
matrix, was observed in the medial temporal lobe. CSPG functions, including regulation of neuronal differentiation
and migration, are highly relevant to the pathophysiology of SZ. CSPGs may exert similar functions in the olfactory
epithelium (OE), a continuously regenerating neural tissue that shows cell and molecular abnormalities in SZ.
We tested the hypothesis that CSPG expression in OE may be altered in SZ. CSPG-positive cells in postmortem
OE from non-psychiatric control (n = 9) and SZ (n = 10) subjects were counted using computer-assisted light
microscopy. ‘Cytoplasmic’ CSPG (c-CSPG) labeling was detected in sustentacular cells and some olfactory recep-
tor neurons (c-CSPG + ORNs), while ‘pericellular’ CSPG (p-CSPG) labeling was found in basal cells and some
ORNs (p-CSPG + ORNs). Dual labeling for CSPG and markers for mature and immature ORNs suggests that c-
CSPG + ORNs correspond to mature ORNs, and p-CSPG + ORNs to immature ORNs. Previous studies in the same
cohort demonstrated that densities of mature ORNs were unaltered (Arnold et al., 2001). In the present study, nu-
merical densities of c-CSPG + ORNs were significantly decreased in SZ (p b 0.025; 99.32% decrease), suggesting a
neurons with respect to basal cells. In this study, we find that the ratio of p-CSPG + ORNs/CSPG + basal cells was
significantlyincreased(p = 0.03)inSZ,whilenumericaldensitychangesofp-CSPG + ORNs(110.71%increase)or
CSPG + basal cells (53.71% decrease), did not reach statistical significance. Together, these results indicate that
CSPG abnormalities are present in the OE of SZ and specifically point to a reduction of CSPG expression in mature
pression may contribute to ORN lineage dysregulation, and olfactory identification abnormalities, observed in SZ.
© 2013 Elsevier B.V. All rights reserved.
Chondroitin sulfate proteoglycans (CSPGs) play a key role in devel-
opmental and adult functions, such as axon guidance, cell adhesion,
differentiation and migration, maturation of synapses and regula-
tion of neurotransmitter receptor availability (Meyer-Puttlitz
et al., 1996; Frischknecht et al., 2009; Maeda et al., 2010). These
functions bear direct relevance to the pathophysiology of schizo-
phrenia (SZ), a disease with a strong neurodevelopmental compo-
nent (e.g. Arnold and Rioux, 2001; Harrison, 2007). Recently,
significant CSPG expression anomalies have been detected in this
disease (Buxbaum et al., 2008; Pantazopoulos et al., 2010; Enwright
et al., 2012; Mauney et al., 2013). In particular, CSPG-enriched
perineuronal nets were decreased in several brain regions, often in asso-
et al., 2010; Enwright et al., 2012; Mauney et al., 2013). Together, these
abnormalities have been postulated to disrupt neurodevelopmental pro-
Schizophrenia Research 150 (2013) 366–372
Abbreviations: BSA, bovine albumin serum; CPZ, chlorpromazine-equivalent; c-CSPG,
cytoplasmic chondroitin sulfate proteoglycan; CSPG, chondroitin sulfate proteoglycan;
GAG, glycosaminoglycan; GAP43, growth associate protein 43; NSCP, neural stem/cell
progenitor; OE, olfactory epithelium; OMP, olfactory marker protein; ORN, olfactory re-
ceptor neuron; PBS–Tx, phosphate buffer–Triton X; p-CSPG, pericellular chondroitin sul-
fate proteoglycan; PMI, postmortem time interval; RE, respiratory epithelium; RPTPz,
receptor tyrosine phosphatase zeta; SCID, Structured Clinical Interview for DSM
Disorders; SZ, schizophrenia; WFA, Wisteria floribunda agglutinin.
⁎ Corresponding author at: MRC3 McLean Hospital — Mailstop 149, 115 Mill Street,
Belmont, MA 02478, USA. Tel.: +1 617 855 3484; fax: +1 617 855 2040.
E-mail address: firstname.lastname@example.org (S. Berretta).
0920-9964/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/schres
CSPG developmental functions are thought to play a key role
throughout life in the olfactory epithelium (OE), a neural structure in
which neuronal differentiation, migration and axon outgrowth occur
robustly throughout life (Clarris et al., 2000; Schwob, 2002). The adult
into mature olfactory receptor neurons (ORNs) (Schwob, 2002). During
the course of their maturation, newly formed ORN axons join odor-
uli (Graziadei, 1973; Yoshihara and Mori, 1997; Beites et al., 2005). In
the OE and olfactory bulb, development-specific patterns of CSPG ex-
pression help position ingrowing olfactory axons in the glomerular
layer and maintain glomerular integrity (Gonzalez and Silver, 1994;
Clarris et al., 2000). CSPG role in regulating brain cell differentiation
(Yanagisawa and Yu, 2007; Purushothaman et al., 2012) suggests that
OE abnormalities observed in SZ are consistent with CSPG dys-
duced adhesion properties and altered cell proliferation in SZ (Feron
of OE cell cycle include lower density of basal cells and increase of post-
mitotic immature ORNs,providingstrongsupportfor a dysregulation of
OE neuronal lineage (Feron et al., 1999; Arnold et al., 2001; Perry et al.,
2002; McCurdy et al., 2006). Taken together, CSPG anomalies in several
key CSPG functions in this structure. We tested the hypothesis that
CSPG expression may be disrupted in the OE of subjects with SZ.
A broad spectrum CSPG histological marker, i.e. Wisteria floribunda
agglutinin (WFA) was used for group comparisons in postmortem
OE tissue; antibodies raised against phosphacan and versican V0/V1,
CSPGs suspected to be involved in SZ and to be expressed in the OE,
were added for normal investigations on cell-specific CSPG distribution
(Clarris et al., 2000; Popp et al., 2003; Buxbaum et al., 2008; Takahashi
et al., 2011) (Pantazopoulos et al., unpublished observations).
2.1. Human subjects and tissue processing
2.1.1. Postmortem and biopsy human OE tissue for normal study
220.127.116.11. Postmortem. A tissue block containing the OE, cribriform plate,
olfactory bulbs, lateral nasal walls and septum from a healthy control
subject (male, 80 years old) was obtained from National Disease
Research Interchange. The tissue block was processed as previously
described (Holbrook et al., 2011). Sections were cut at 10 μm and
mounted on super frost plus slides.
18.104.22.168. Biopsy. Tissue samples containing the OE from two healthy con-
trols (males — 23 and 41 years of age) were obtained by biopsy under
local anesthesia. Medical history, current medical status and absence of
psychiatric disorders (Structured Clinical Interview for DSM Disorders,
and biopsy were approved by the Institutional Review Boards of McLean
Hospital and Massachusetts Eye and Ear Infirmary. Subjects provided
written informed consent prior to their inclusion in the study. Biopsy
samples were postfixed in 4% paraformaldehyde for 1 h, cryoprotected
in 30% sucrose overnight and sectioned on a cryostat (10 μm).
2.1.2. Postmortem human OE tissue for group comparisons
PostmortemOEtissuewascollected from 10 chronicSZpatientsand
9 age- and sex-matched non-psychiatric controls (Table 1). All subjects
vania and were clinically assessed and diagnosed according to DSM-IV
criteria by research psychiatrists of the University of Pennsylvania's
Schizophrenia Mental Health Clinical Research Center, Philadelphia, as
previously described (Arnold et al., 2001). This involved a standardized
medical record review of demographic variables, presenting and sub-
sequent symptoms, treatment history, medical history, caregivers'
interview and laboratory and neuroimaging findings. Based on all
information, diagnoses and inclusion were established by research
team consensus. Non-psychiatric controls were obtained through the
University of Pennsylvania's Alzheimer Disease Core Center. Review of
clinical histories found no evidence of prior major psychiatric or neuro-
logical illnesses. Gross and microscopic diagnostic neuropathologic
examinations of multiple cortical and subcortical regions revealed no
cular accidents in anyof thesubjects included in this cohort. At autopsy,
the nasalepithelium, bony septae, andcontiguous cribriform plate were
removed en bloc and processed as above (Arnold et al., 2001).
2.2. Histochemistry and immunohistochemistry
2.2.1. Single immunohistochemistry/histochemistry for CSPGs
Tissue sections were blocked in 2% bovine albumin serum (BSA).
For immunohistochemistry, sections were incubated for 48 h in
chlorpromazine-equivalent (CPZ) dose (expressed in average mg/day during the last month of life); Dx, diagnosis; PMI, postmortem interval (hours).
Dx AgeSexPMIBrain weight CPZ last month of lifeAge at onsetYears of illness
75.4 +/− 4.2
72.4 +/− 15.8
5 M/5 F
5 M/4 F
11.8 +/− 3.5
10.7 +/− 8.3
1178.6 +/− 85.0
1289.9 +/− 203.3
350 +/− 382.4
24.11 +/− 5.4
50.89 +/− 8.6
H. Pantazopoulos et al. / Schizophrenia Research 150 (2013) 366–372
primary antibodies (see below), then for 2 h at room temperature in
biotinylated secondary antibody (1:500 goat anti-rabbit, Vector Labs
BA-1000, lot x0212); for histochemistry, sections were incubated in
Wisteria floribunda agglutinin (WFA; see also Supplementary material)
in 1% BSA for 24 h at 4 °C. For both immunocytochemistry and histo-
chemistry, sections were then incubated in streptavidin (1:5000;
Invitrogen 434323, lot 830799A). Nickel-enhanced diaminobenzidine/
peroxidase reaction (0.02% diaminobenzidine (Sigma-Aldrich, St. Louis,
MO), 0.08% nickel-sulfate, 0.006% hydrogen peroxide) was used to visu-
phosphate buffer/saline with 0.5% Triton X (PBS–Tx). Each step was
followed by washes in the same solution. Antibodies: receptor tyrosine
phosphate zeta (RPTPz)/phosphacan (1:2000; Abcam ab126497, lot#
GR79986; immunogen was a synthetic peptide corresponding to
Fig. 1. Patterns of CSPG expression in the normal human OE. In normal human OE, CSPG labeling using the broad spectrum marker WFA shows two distinct labeling patterns in OE cells:
patterns.Highresolution fluorescent confocal imagesofWFAlabeledcells (green) and DAPI labelednuclei(blue) confirmthis pattern (C,D). InE–G,dualfluorescence labelingcombining
WFA with GAP43, prevalently expressed in immature ORNs, shows that p-CSPG + ORNs correspond to GAP43 + ORNs (arrow heads). In contrast, dual fluorescence labeling combining
WFA with OMP, which typically labels mature ORNs, shows that c-CSPG + ORNs correspond to OMP + ORNs (H–J; arrows). Together, these findings indicate that i) basal cells present
withapericellularpatternofCSPG/WFA;ii)immatureORNsmaintainthep-CSPGexpressionpattern;andiii)mature ORNsappearto transitionto aprevalentcytoplasmicpatternof CSPG
expression. Scale bar, 50 μm.
H. Pantazopoulos et al. / Schizophrenia Research 150 (2013) 366–372
amino acids 831–978 of the human RPTPz/phosphacan sequence —
made in rabbit); glycosaminoglycan beta (GAGbeta)/versican V0/V1
(1:8000; gift from M.T. Dours-Zimmermann and D.R. Zimmermann,
Univ. of Zurich, Switzerland – identifies the C-terminus amino acids
2646–3088 of human versican – made in rabbit) reported to label
both the V0 and V1 versican isoforms (Dours-Zimmermann and
Zimmermann, 1994). Biotinylated WFA (1:1000; Vector Labs, #B-
1355, lot W0103, Burlingame, CA).
2.2.2. Dual antigen immunofluorescence
Double labeling for mature ORNs and CSPGs was carried out using a
primaryantibodyraised ingoatagainst olfactorymarker protein (OMP)
(1:12,000, Wako, #544-10001, lot 1UP-1001, Richmond VA) in combi-
nation with biotinylated WFA (1:2000, Vector Labs). Immature ORNs
were labeled using a primary antibody raised in mouse against growth
associated protein 43 (GAP-43) (1:500, Millipore, clone 9-1E12
#MAB347, lot NG1869850, Temecula, CA), in combination with WFA.
After blockingin 2% BSA, sections were incubated in primary antibodies
with 2% BSA at 4 °C for 48 h, followed by a combination of either Alexa
Fluor donkey anti-goat 594 (1:250, Invitrogen #A11058, lot 1003216,
Grand Island, NY) and Alexa Fluor Streptavidin 488 (1:3000, Invitrogen
#S-11223), or Alexa Fluor donkey anti-mouse 594 (1:250, Invitrogen
A21203, lot 987237) and Streptavidin 488 (1:3000, Invitrogen #S-
long Gold anti-fade mounting media with DAPI (Invitrogen P-36931).
Solutions for all the steps above were made in PBS with 0.5% Triton X.
2.3. Data collection
2.3.1. CSPG-positive cell phenotype in normal human OE
Sections from the OE (3–4 sections per subject), dual labeled for
CSPGs (WFA) in combination with OMP or GAP43 from 3 control sub-
jects (two biopsy and one postmortem samples) were examined using
a Zeiss Axioskop II plus fluorescence imaging system, interfaced with
Stereo-Investigator 6.0 (MicroBrightField, Inc., Williston, VT). OE areas
in each section, interspersed among respiratory epithelium (RE)
patches, were identified according to established structural and
cytoarchitectonic criteria (Moran et al., 1982; Morrison and Costanzo,
1990; Holbrook et al., 2011) (compare Figs. 1A, B to 3A). OE areas
were scanned at 40× throughout the extent of the x, y, and z axes to
count CSPG-positive (CSPG+) cells with cytoplasmic or pericellular
labeling, and their expression, or lack thereof, of OMP or GAP43. Cy-
toplasmic and pericellular WFA labeling was confirmed on a subset
of sections at 63× using a Leica TCS SP8 confocal imaging system
(z-axis resolution: 1 μm through the extent of the tissue section;
Fig. 1C, D). Cell morphology and immunolabeling distribution of
versican V0/V1 and RPTPz/phosphacan (Fig. 2) were analyzed
under 40× magnification.
2.3.2. CSPG-positive cells in OE and RE — group comparison
Slides were coded for analysis blind to diagnosis. Computer-assisted
light microscopy interfaced with stereology quantification software
(see above) was used for data collection. Two to four sections per sub-
ject were available for quantification. The borders between the OE and
RE areas were drawn as described above. Sections were scanned
through the extent of the x, y, and z axes within each area under 40×
magnification and all CSPG-labeled cells were counted. Intra-rater
(H.P.) reliability of at least 95% was established before the actual quan-
tification process begun and assessed on a regular basis throughout the
quantification process. Morphologically distinct CSPG+ cell categories
were identified in the OE (see Moran et al., 1982; Morrison and
Costanzo, 1990; Holbrook et al., 2011). CSPG + ORNs were further
subdivided in two groups according to their pericellular (p-CSPG+) or
cytoplasmic (c-CSPG+) labeling pattern (see Fig. 1 and Section 3: Re-
sults). In RE, CSPG+ cells corresponded almost exclusively to basal
cells (Fig. 4A).
2.4. Statistical analysis
For the purpose of group comparisons, numerical densities of
CSPG+ cells for each subject were the main outcome measure (sum
of labeled cells divided by the sum of the areas of OE or RE). Total num-
bers of cells were not calculated because the anatomical characteristics
of the OE, which is constituted of islands intermingled with RE
(Holbrook et al., 2011), do not allow reliable total volume estimates.
Statistical significance of differences between groups relative to the
main outcome measures was assessed using a stepwise linear regression
process. A logarithmic transformation was uniformly applied to all raw
values because the data was not normally distributed. Statistical analyses
were performed using JMP v5.0.1a (SAS Institute Inc., Cary, NC). Age,
of antipsychotic drugs taken during the last month of life, expressed as
chlorpromazine-equivalent (CPZ) dose, were tested systematically for
their effects on numerical densities and included in the model if they
significantly improved the model goodness-of-fit (Table 1). In addition,
potential effects of exposure to antipsychotics, as well as age at the
onset of the disease and duration of the illness, were tested in separate
linear correlation analyses (see also Supplementary information).
3.1. CSPG expression in normal human OE
In the OE, two clearly distinguishable patterns of CSPG labeling,
pericellular and intracellular, were detected. Sustentacular cells and
ing in a granular pattern within the cytoplasm, often more intense near
thenucleus (Fig. 1H–J). Incontrast, basal cells and a secondsubgroup of
Fig. 2. Versican V0/V1 and phosphacan expression in normal human OE. Light microscopy photomicrographs of normal human OE stained for versican V0/V1 and phosphacan. Versican
V0/V1 showed cytoplasmic expression (arrows) similar to that detected in mature ORNs. Phosphacan predominantly showed pericellular expression patterns (arrow heads) strongly re-
sembling that observed with WFA in immature ORNs and basal cells.
H. Pantazopoulos et al. / Schizophrenia Research 150 (2013) 366–372
ORNs presented with pericellular CSPG labeling (Fig. 1A–D). The simi-
larity of CSPG labeling pattern in basal cells and a subset of ORNs raised
the possibility that the latter may correspond to immature, GAP43-
positive, ORNs. Dual immunofluorescence labeling of normal OE
CSPG + ORNs expressed GAP43 (Fig. 1E–G), a protein prevalently
contained in immature ORNs (Verhaagen et al., 1989; Hahn et al.,
2005), while only 1.9% of c-CSPG + ORN was GAP43-positive. Con-
versely 91.2% of c-CSPG +ORN expressed OMP (Fig. 1H–J), a marker
for mature ORNs (Farbman and Margolis, 1980; Hahn et al., 2005),
while 15.0% of p-CSPG + ORNs were found to be OMP-positive. Finally,
we tested whether the two different patterns of CSPG cellular distribu-
tion may reflect expression of distinct CSPGs. Immunolabeling for
RPTPz/phosphacan showed predominant pericellular ORN and basal
exclusively with intracellular ORN labeling (Fig. 2A).
this possibility.Thelargemajority(89.4%) of
3.2. CSPG-positive cells in the OE and RE of subjects with SZ
In the OE, numerical densities of c-CSPG + ORNs were significantly
decreased in subjects with SZ compared to control subjects (step-wise
linear regression analysis; p b 0.025; 99.32% decrease; Fig. 3A). Numer-
ical density increases of p-CSPG + ORNs (110.71%) and decreases of
icance (Fig. 3A). The ratio of p-CSPG + ORNs/CSPG+ basal cells signifi-
cantly increased in subjects with SZ (p = 0.038, t = 2.31). Ratios of
c-CSPG + ORNs/CSPG+ basal cells and p-CSPG + ORNs/c-CSPG +
ORNs were not altered. Numerical densities of CSPG+ sustentacular
cells were not altered in subjects with SZ (Fig. 3A). In the RE, numer-
ical densities of CSPG+ basal cells were similar in the two groups
None of potential confounding variables tested with stepwise linear
regression models showed significant effects. Antipsychotic exposure
(Fig. 3B) and other potential disease-related factors, such as age at
ear correlation analysis (see also Supplementary information). A posi-
tive correlation of c-CSPG + ORNs with years of illness suggests that
decreases of these cells are not due to non-specific effects of chronic
These findings show, to our knowledge for the first time, that CSPGs
are expressed in mature and immature ORNs, sustentacular cells and
basal cells in the adult human OE, with a distinct, cell specific, localiza-
tion pattern. Furthermore, our results represent the first evidence for
CSPG abnormalities in the OE of SZ subjects. Thus, we show that these
abnormalities encompass not only distinct CNS regions but also
Fig. 4.Numerical densities of CSPG+ cells were not significantlydifferent inthe RE of SZsubjects.CSPG+ cells inREcorrespondedalmost exclusivelyto basal cells.In(A), examplesof RE
histological stained for CSPGs (WFA) show dense black labeling. Scale bar, 50 μm. (B) No differences of numerical density of CSPG+ basal cells and RE cells were observed between SZ
and non-psychiatric control subjects. Statistical analyses were performed using stepwise linear regression models. Scatter plots show the mean (black line) and 95% confidence intervals
Fig. 3. Densities of c-CSPG + ORNs are decreased in the OE of SZ subjects. Numerical
densities of c-CSPG + ORNs were significantlydecreased(p = 0.02)intheOEof subjects
withSZ,ascomparedtonon-psychiatriccontrolsubjects(A).Increasesofp-CSPG + ORNs
and decreases of CSPG+ basal cells did not reach statistical significance. Significance
valuesare derived from stepwise linear regression models testing potential effects of con-
found variables. Scatter plots show the mean (black line) and 95% confidence intervals
(gray lines). No significant correlation was observed between c-CSPG + ORNs and chlor-
promazine normalized (CPZ) dose of antipsychotics (B).
H. Pantazopoulos et al. / Schizophrenia Research 150 (2013) 366–372
peripheral sensory structures, potentially contributing to functional
sensory impairment (Moberg et al., 1999; Turetsky et al., 2009). These
abnormalities prevalently affect cells putatively corresponding to ma-
ture ORNs. This finding suggests that, in addition to CSPG+ glial cell
and perineuronal net anomalies previously detected in the CNS, CSPG
pathology in SZ may also affect maturing neurons, disrupting ORN matu-
ration and olfactory system connectivity and, potentially, brain develop-
ment. Below we discuss the possibility that these abnormalities may
reflect a dysregulation of CSPG expression in the OE of subjects with SZ,
and put forth the hypothesis that they may contribute to a disruption of
cell lineage in OE in SZ (Arnold et al., 2001).
4.1. CSPG expression in normal human OE
The observed pericellular pattern of CSPG labeling (WFA and
phosphacan) in basal cells is highly reminiscent of that of CSPG expres-
sion in neural stem/cell progenitors (NSCPs) in stem cell niches of the
embryonic and adult brain (Ito et al., 2005; von Holst et al., 2006). In
these cells, surface expression of CSPGs was found to be essential for
mine maturation, differentiation and migration (von Holst et al., 2006;
Sirko et al., 2010). We suggest that the p-CSPG labeling in OE basal
cells may mediate similar functions, such as proliferation of basal cells
and their differentiation into immature ORNs. In mature ORNs, WFA/
CSPG expression was instead found predominantly in the cytoplasm.
Versican, which is also expressed in the cytoplasm of neurons in the
brain,showeda similarpattern. Thus,ourresultssuggestthatORNmat-
uration involves a transition of CSPG type and distribution: from
pericellular phosphacan expression to versican V0/V1 expression in
the cytoplasm. The functional implications of the transition between
phosphacan and versican can only be inferred on the basis of current
knowledge on brain CSPG functions, because their expression in the
adult OE has not been reported before. We suggest that this transition
may involve a shift in the ORN interactions with other cells and the ex-
tracellular environment (Rhodes and Fawcett, 2004; Carulli et al., 2005;
Dityatev et al., 2010). While electron microscopy will determine the ul-
trastructural localization of CSPGs in OE cells, for the purpose of this
study, we use the term ‘pericellular’ to indicate CSPG labeling at the
cell periphery, as clearly distinct from the CSPG labeling pattern detect-
ed in mature ORNs.
4.2. CSPG abnormalities in SZ
Comparisonsbetweennon-psychiatric control and SZ subjects show
a significant decrease of c-CSPG + ORN, putatively corresponding to
mature (OMP+) ORNs. Previous results from a largely overlapping set
crease, of OMP + ORNs in the OE of subjects with SZ. Together, these
findings suggest that decreased c-CSPG + ORN densities reflectreduced
CSPG expression in mature ORNs, rather than a decrease of mature
ORNs. This reduction may be due to decreased synthesis and/or failure
of these cells to fully transition to a mature pattern of CSPG expression.
A significant increase of the p-CSPG + ORN/CSPG+ basal cell ratio,
consistent with increased ratio of immature ORN/basal cells reported
by Arnold et al. (2001), supports the latter possibility and may offer a
clue on a potential contributing mechanism. As mentioned above, cell
surface CSPG expression in NSCPs is needed for cell differentiation and
maturation, mediated by interactions with growth factors (Sirko et al.,
2010). Altered p-CSPG + ORN/CSPG+ basal cell ratio in SZ may
reflect a disruption of this process, and thus ORN's failure to transition
from p-CSPG to a mature c-CSPG expression patterns. Overall, these
findings add to evidence for a disruption of OE cell lineage in SZ and
raise the possibility that abnormal CSPG expression may contribute to
such disruption. It should be recognized, however, that while the dis-
crepancy between c-CSPG-ORNs decreases in this study and normal
densityof OMP + ORNs previouslyreported inthesamesubjectcohort
results from the 2001 study do not allow to unambiguously interpret in-
creased ratios of pCSPG + ORNs/CSPG+basalcells inSZas altered CSPG
expression in immature ORNs and basal cells.
On a speculative level, we postulate that aberrant CSPG expression
inORNsmaydisruptORN axonaloutgrowthand guidance,assuggested
by preclinical studies (Clarris et al., 2000). CSPGs, and versican in
particular, promote axon outgrowth (Wu et al., 2004; Maeda, 2010;
Klausmeyer et al., 2011). CSPGs have been shown to play a key role
inguidingORN axonsacross thelaminapropriaandthroughthelamina
cribrosa, and eventually forming bundles segregated by odor specificity
and reaching odor specific glomeruli in the olfactory bulb (Gonzalez
and Silver, 1994; Yoshihara and Mori, 1997; Tisay and Key, 1999;
Belluscio and Katz, 2001; Hayar et al., 2004). It is conceivable that
and guidance resulting in mismatched and/or disorganized synaptic
formation between the OE and the olfactory bulb. This phenomenon
may contribute robust olfactory deficits detected in subjects with SZ
(Kopala et al., 1993; Moberg et al., 1999; Brewer et al., 2001; Atanasova
et al., 2008).
Caution in interpreting these results is necessary given the small
sample size (9normal control and 10 SZ subjects for thegroup compar-
ison study). Although the sample size in this study is small, it is compa-
rable to a similar published report using postmortem OE samples from
SZ (13) and control (10) subjects (Arnold et al., 2001).
In summary, these results show CSPG abnormalities in the OE of SZ
subjects. Given the role that CSPGs play in cell differentiation and ORN
neurite outgrowth and axon guidance, altered CSPG expression may
contribute to cell lineage and olfactory identification abnormalities de-
tected in SZ (Arnold et al., 2001; Turetsky et al., 2008). These results
vous system and detectable in tissue, such as the OE, that can be
harvested by biopsy. Future studies will use this approach to test mech-
anisms of CSPG abnormalities in SZ and their association with specific
core clinical aspects of this disorder.
Role of funding source
This work was funded by the National Institutes of Health — NIMH.
R01-MH091348 to Sabina Berretta, M.D.
Harry Pantazopoulos ran the immunocytochemistry, histochemistry, and data collec-
tion for group comparisons, performed the statistical analysis and wrote the first draft of
Anne Boyer-Boiteau was responsible for the study in normal OE, including single and
double immunocytochemistry, data collection and confocal microscopy, and contributed
to the preparation of the manuscript.
ed technical advice on tissue preparation and immunocytochemistry and contributed to
the preparation of the manuscript.
Woochan Jang contributed to aspects of tissue processing (biopsy) and immunocyto-
chemistry and to the preparation of the manuscript.
Chang-Gyu Hahn contributed to data analysis and preparation of the manuscript.
Steven E. Arnold provided the tissue for group comparison and contributed to the
design of the study and preparation of the manuscript.
uted to data analysis and took the lead on the preparation of the manuscript.
All authors have approved the final manuscript.
Conflict of interest
Harry Pantazopoulos: No conflict.
Anne Boyer-Boiteau: No conflict.
Eric H. Holbrook: No conflict.
Woochan Jang: No conflict.
Chang-Gyu Hahn: No conflict.
Steven E. Arnold: No conflict.
Sabina Berretta: No conflict.
H. Pantazopoulos et al. / Schizophrenia Research 150 (2013) 366–372
The authors are grateful to M.T. Dours-Zimmermann and D.R. Zimmermann, Univ. of
Zurich, Switzerland for their generous gift of GAGbeta/versican V0V1antibody and to
NIMH (R01MH091348 to SB) for funding this study.
Appendix A. Supplementary data
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