www.frontiersin.org May 2011 | Volume 2 | Article 27 | 1
Original research article
published: 13 May 2011
markers of oxidative stress are elevated in the orbitofrontal cortex
in post-mortem samples of ASC patients (Sajdel-Sulkowska et al.,
2011). This region is thus a likely candidate for an underlying cel-
Nuclear factor kappa-light-chain-enhancer of activated B cells
(NF-κB) is a protein that controls transcription of DNA and is
found in almost all cell types (Perkins, 2004). It mediates cellular
response to external stressors and is central to the regulation of
immune responses by inducing the expression of inflammatory
cytokines and chemokines and, in turn, being induced by them
(Pahl, 1999; Perkins, 2004). This establishes a positive feedback
mechanism (Larsson et al., 2005) which, when NF-κB becomes
aberrantly active, has the potential to produce chronic or excessive
inflammation associated with several inflammatory diseases (Pahl,
1999). Furthermore, post-mortem studies suggest that NF-κB plays
a key role in Alzheimer’s disease (Akiyama et al., 1994) and its pos-
sible treatment (Paris et al., 2007), Parkinson’s disease (Block and
Hong, 2005) and multiple sclerosis (Glass et al., 2010).
Microglia mediate the immune responses of the central nervous
system acting to remove extracellular debris with a similar func-
tion to macrophages. Microglial cells have been associated with
Autism spectrum condition (ASC) is a life-long neurodevelop-
mental condition characterized by a triad of impairments in social
skills, verbal communication, and behavior (Rapin, 1997; Lord
et al., 2000). Cognitively, ASC is described as a disorder involving
fundamental deficits in central coherence (Frith, 1989), executive
function (Ornitz et al., 1993), theory of mind (Baron-Cohen et al.,
1985), and empathizing (Baron-Cohen, 2002). Continuing inves-
tigations for a neurobiological basis for ASC support the view that
genetic, environmental, neurological, and immunological factors
contribute to its etiology (Neuhaus et al., 2010). In particular, there
is evidence to suggest an association between ASC and neuroin-
flammation in anterior regions of the neocortex (Pardo et al., 2005;
Vargas et al., 2005; Zimmerman et al., 2005), and areas relating to
cognitive function appear to be affected by inflammation resulting
from activation of microglia and astrocytes (Anderson et al., 2008).
In vivo measurements of structural brain changes with magnetic
resonance imaging have detected gray matter loss in the orbitofron-
tal cortex (Hardan et al., 2006; Girgis et al., 2007) and impairment
of cognitive functions mediated by the orbitofrontal–amygdala
circuit (Loveland et al., 2008) in patients with ASC. Furthermore,
Aberrant NF-kappaB expression in autism spectrum condition:
a mechanism for neuroinflammation
Adam M. H. Young1,2, Elaine Campbell1, Sarah Lynch1, John Suckling3* and Simon J. Powis1
1 Bute Medical School, University of St. Andrews, Fife, Scotland, UK
2 Autism Research Unit, Department of Psychiatry, University of Cambridge, Cambridge, UK
3 Department of Psychiatry, University of Cambridge, Cambridge, UK
Autism spectrum condition (ASC) is recognized as having an inflammatory component. Post-
mortem brain samples from patients with ASC display neuroglial activation and inflammatory
markers in cerebrospinal fluid, although little is known about the underlying molecular
mechanisms. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein
found in almost all cell types and mediates regulation of immune response by inducing the
expression of inflammatory cytokines and chemokines, establishing a feedback mechanism
that can produce chronic or excessive inflammation. This article describes immunodetection and
immunofluorescence measurements of NF-κB in human post-mortem samples of orbitofrontal
cortex tissue donated to two independent centers: London Brain Bank, Kings College London, UK
(ASC: n = 3, controls: n = 4) and Autism Tissue Program, Harvard Brain Bank, USA (ASC: n = 6,
controls: n = 5). The hypothesis was that concentrations of NF-κB would be elevated, especially in
activated microglia in ASC, and pH would be concomitantly reduced (i.e., acidification). Neurons,
astrocytes, and microglia all demonstrated increased extranuclear and nuclear translocated
NF-κB p65 expression in brain tissue from ASC donors relative to samples from matched
controls. These between-groups differences were increased in astrocytes and microglia relative
to neurons, but particularly pronounced for highly mature microglia. Measurement of pH in
homogenized samples demonstrated a 0.98-unit difference in means and a strong (F = 98.3;
p = 0.00018) linear relationship to the expression of nuclear translocated NF-κB in mature
microglia. Acridine orange staining localized pH reductions to lysosomal compartments. In
summary, NF-κB is aberrantly expressed in orbitofrontal cortex in patients with ASC, as part of
a putative molecular cascade leading to inflammation, especially of resident immune cells in
brain regions associated with the behavioral and clinical symptoms of ASC.
Keywords: NF-κB, autism spectrum condition, brain, inflammation, orbitofrontal cortex, pH
Lin He, Shanghai Jiao Tong University,
Gráinne McAlonan, University of Hong
Kong, Hong Kong
Elizabeth Maria Sajdel-Sulkowska,
Harvard Medical School, Brigham and
Women’s Hospital, USA
John Suckling, Department of
Psychiatry, University of Cambridge,
Herchel Smith Building, Robinson Way,
Cambridge CB2 0SZ, UK.
Frontiers in Psychiatry | Molecular Psychiatry
May 2011 | Volume 2 | Article 27 | 2
Young et al. Aberrant NF-kappaB expression in ASC
NF-κB, and thus its potential for nuclear translocation. The
expression of active NF-κB translocated to the cell nuclei was
then measured directly, where it binds to DNA and transcribes
proteins that result in the production of cytokines as part of an
Confirmation of the immunodetection and immunofluores-
cence results in neurons and mature microglia was sought from
an independent source of micro-array tissue slides donated from
ASC patient and control groups.
Finally, pH was measured in homogenized tissue and com-
pared to the corresponding intracellular NF-κB p65 expression
from Western immunodetection. Acridine orange staining allowed
measurements of pH localized to lysosomes.
MaterIals and Methods
The UK cohort consisted of seven samples of fixed orbitofrontal
cortex sections from four control and three ASC donors obtained
from the Medical Research Council’s London Brain Bank (Institute
of Psychiatry, King’s College London). Samples were age, sex, and
post-mortem interval (PMI) matched (Table 1). Protein extrac-
tion from formalin fixed tissue was performed according to Shi
et al. (2006), where tissue sections were placed in 50 μl of 20 mM
Tris–HCl buffer pH 8 with 2% SDS plus pepstatin 10 μg/ml and
heated to remove formalin cross-links.
Samples of the US cohort were sections from six ASC patients
and five age, sex, and PMI matched control donors obtained as
tissue micro-arrays (Eberhart et al., 2006) from the Autism Tissue
Program (Harvard Brain Tissue Resource Center, Boston). One
patient had a diagnosis of Rett syndrome, a neurodevelopmental
brain inflammation (Liu and Hong, 2003; Barger, 2005; Kim and
Joh, 2006) and pro-inflammatory treatments of microglia acidify
cellular lysosomes to a pH ∼5 (Majumdar et al., 2007). Preliminary
characterization of this mechanism implicates activation of protein
kinase A (PKA) and the activity of chloride channels (Majumdar
et al., 2007). The transcriptional activity of NF-κB is stimulated
upon phosphorylation of its p65 subunit on serine 276 by PKA
(Zhong et al., 1998) and in turn PKA is a downstream target of the
transcription factor (Kaltschmidt et al., 2006). With this in mind we
postulated that an association may exist between the transcription
factor and lysosomal acidity.
This article describes measurement of NF-κB p65 expression
levels and pH in post-mortem samples of orbitofrontal cortex from
patients with a diagnosis of ASC and control samples from people
healthy at the time of death. We hypothesized that concentrations
of NF-κB would be elevated in patients and pH would be con-
comitantly reduced (i.e., acidification), providing evidence for a
neuroinflammatory component to ASC.
This hypothesis was initially tested by Western immunodetec-
tion of post-mortem brain tissue to measure overall, nuclear and
cytosolic NF-κB expression.
Investigations were then focused upon microglial cells due to
their role in pro-inflammatory response, as these most strongly
mediate aberrant expression of NF-κB. Antigen retrieval and
immunofluorescence techniques were used to identify the dif-
ferential concentrations of intracellular NF-κB in neurons, astro-
cytes, microglia, and highly activated (i.e., mature or functional)
microglia. Immunoreactivity measurements were initially carried
out to determine the concentration of NF-κB in the cytoplasm
of each cell type as an indication of the availability of inactive
Table 1 | Demographic information on tissue donors for the UK (London Brain Bank) and US (Autism Tissue Program/Harvard Brain Tissue Resource
Sample ID Autopsy no. Diagnosis Age (years) Sex Cause of death PMI* (hours) Cohort
Chronic obstructive pulmonary disease
Accident; multiple injuries
1Epilepsy treated with carbamazepine and vigabatrin.
2Carcinoma treated with zuclopenthixol, fluconazole, ranitidine, omeprazole, prednisolone, and morphine.
www.frontiersin.org May 2011 | Volume 2 | Article 27 | 3
Young et al. Aberrant NF-kappaB expression in ASC
microglia (Hughes et al., 2003). This was followed by a final 5 min
wash and slides were mounted with 4′,6-diamidino-2-phenylin-
dole (DAPI) with Vectashield (Vector Labs Ltd, UK) to identify
One hundred cells of each type (neurons, astrocytes, and micro-
glia) from each sample were selected at random, blinded from
group (i.e., ASC or control). Cells were graded for immunoreac-
tivity according to the intensity of antibody signal within the cell
on an integer scale of 0–3 (Schmidt and Bankole, 1965). For each
cell type, the percentage of cells with weak (scale 1) and strong
(scale 3) intensity was calculated.
Antigen positive cells were also counted in each sample to quan-
tify the intensity of anti-p65 signal in the nucleus, providing a
measurement of nuclear translocation of NF-κB p65 and thus the
active state of the molecule within each cell type.
MeasureMent of ph
Equal volumes of neural tissue were homogenized using a mortar
and pestle in 10 volumes of deionized water at 4°C. The pH of the
homogenate was measured using a MeterLab PHM201 pH meter
(Radiometer Analytical, Villeurbanne Cedex, France) calibrated
with two standards for pH 4 and 7. Measurements were made eight
times over 4 days and averaged to yield a final value.
Using the protocol described by Morgan and Galione (2007) and
Lee et al. (1983), the pH of lysosomes was measured. Tissue sections
were simultaneously loaded with 10 μM acridine orange and 1 μM
Lysotracker Red DND-99 for 15–20 min at room temperature, at
which time the fluorescence had reached equilibrium; that is, the
dyes were present throughout the rest of the experiment. Acridine
orange responds rapidly and profoundly to changes in pH, whereas
Lysotracker Red responds only relatively slowly and remains essen-
tially fixed throughout the experiment. Results are expressed as the
ratios of the acridine orange/Lysotracker Red signals such that an
increase in the ratio reflects an increase in pH.
Small sample sizes precluded valid formal between-group (i.e., ASC
vs. control) statistical tests. Nevertheless, in many cases there was
no overlap in the values obtained for each group. Unless indicated,
all data are expressed as mean ± standard error across the samples.
Statistical testing of within-group correlation was undertaken using
SPSS (v17, SPSS Inc.), with the level for significance set at p < 0.05.
expressIon of nf-κB p65 In neural tIssue: uK cohort
Western immunodetection of neural tissue samples analyzed for
overall NF-κB p65 expression is shown in Figure 1A. Densitometry
demonstrated a 2.9-fold increase of NF-κB p65 expression in ASC
samples; Figure 1B.
Nuclear translocation of NF-κB p65 is predominately associ-
ated with the activation of the transcription factor. Separated tis-
sue lysates were used to determine the subcellular location of p65
expression. In control tissue NF-κB p65 was mainly located within
the cellular cytoplasm, whereas in tissue samples from ASC patients
the expression was predominately within the nucleus. Western
immunodetection of neural tissues analyzed for overall NF-κB
p65 expression for the UK cohort is shown in Figure 1C.
disorder classified as an ASC. The age-at-death of donors
was considerably younger (5–11 years) than the UK cohort
(20 –79 years; Table 1).
Tissue samples from the UK cohort were processed for nuclear and
cytosolic extraction using two separation buffers. The tissue was
homogenized in 10 volumes of buffer 1 (Tris 10 mM, NaH2PO4
20 mM, EDTA 1 mM, pH 7.8 PMSF 0.1 mM, pepstatin 10 μg/ml,
and leupeptin 10 μg/ml). Homogenate was incubated for 20 min
and osmolarity restored by adding 1/20 volume of KCl 2.4 M,
1/40 volume of NaCl 1.2 M, 1/5 volume sucrose 1.25 M. Samples
were spun for 5 min at 3,500 rpm, supernatant removed and pel-
let resuspended on 0.6 M sucrose and spun for a further 10 min
at 10,000 rpm.
Subsequently, the supernatant was diluted in buffer 2 (imidazole
30 mM, KCl 120 mM, NaCl 30 mM, NaH2PO4, sucrose 250 mM pH
6.8, protease inhibitors pepstatin 10 μg/ml and leupeptin 10 μg/
ml) and spun again at 3,500 rpm for 15 min. The resultant pellets
contained the remaining nuclear proteins, and the supernatants
the cytosolic proteins.
Protein samples run on SDS-polyacrylamide gels were elec-
troblotted to nitrocellulose membranes (Schleicher & Schuell
Bioscience GmbH, Germany), blocked with 5% non-fat dry milk
in phosphate buffered saline with 0.1% Tween-20 (PBST), and
probed with a 1:1,000 dilution of anti-p65 antibody (Santa Cruz
Biotechnology). PBST washed membranes were then incubated
with HRP-conjugated goat anti-rabbit antisera (Sigma Ltd, UK),
and developed with enhanced chemiluminescence reagents (Pierce
Ltd, UK). Signal was detected using a LAS 3000 image analyzer
(Fujifilm, Japan) and bands quantified using ImageJ software.
Tissue samples were fixed in formalin and embedded in paraffin
blocks. Slides were deparaffinized in xylene three times, each for
5 min, then hydrated gradually through graded alcohols: washed
in 100% ethanol twice for 10 min each, then 95% ethanol twice
for 10 min each and finally washed in deionized water for 1 min
with stirring. Sections were cut 5 μm thick and mounted onto these
Antigen retrieval was carried out in a pressure microwave where
slides were covered in 10 mM sodium citrate buffer pH 6.0. After
cooling for 20 min, sections were blocked in 10% normal goat block
for 15 min and placed in anti-p65 NF-κB antibody 1:200 (Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C.
Sections were washed continuously for 5 min and placed in HRP-
conjugated goat anti-rabbit antisera 1:200 (Molecular Probes Inc.,
Eugene, OR, USA) for 30 min at room temperature.
Sections were then stained with either: anti-Beta III Tubulin
(1:10,000), a microtubule element antibody targeted exclusively in
neurons; anti-GFAP (1:250), an intermediate filament (IF) protein
antibody specific for astrocytes; anti-CD11b (1:250), a complement
component 3-receptor 3-subunit antibody sensitive to microglia
(Ford et al., 1995; Becher and Antel, 1996); or anti-CD11c (1:250),
a type I transmembrane protein antibody found on highly activated
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May 2011 | Volume 2 | Article 27 | 4
Young et al. Aberrant NF-kappaB expression in ASC
the control group had the majority of fluorescence scores in
the range 0–1 (69%; Table 2). There was an increase in nuclear
translocation of NF-κB p65 in neurons, within tissue from ASC
patients (21.67 ± 1.50%) compared to controls (17.00 ± 1.41%;
Figure 2A). Examples of CD11c, p65, and DAPI staining are
shown Figure 3D.
Between-group differences in the immunoreactivity of astro-
cytes stained by anti-GFAP were observed with 80% of cells from
ASC samples scoring 2–3 compared to 56% of cells from con-
trol samples scoring 0–1; Table 2. Differences in nuclear NF-κB
p65 expression between ASC and control tissue samples were
observed. On average 88.00 ± 4.00% of astrocytes in tissue from
ASC patients demonstrated nuclear localization of the tran-
scription factor compared to 33.00 ± 3.16% in control samples;
expressIon of nf-κB p65 In neurons, astrocytes, and
MIcroglIa: uK cohort
The immunoreactivity intensity from neurons stained by anti-
Beta III Tubulin in tissue from ASC patients predominately
scored in the range 2–3 (78%), whilst neurons in tissue from
Table 2 | Immunoreactivities of each cell type on an integer scale of 0 (weakest) to 3 (strongest) intensities.
Cell type Control samples ASC samples
0 1 2 3 0 1 2 3
Microglia CD11b+ (UK)
Microglia CD11c+ (UK)
Microglia CD11c+ (US)
27 .30 (20.30)
28.30 (7 .09)
56.67 (7 .44)
Values are mean (across samples, with standard deviation) percentage of cells demonstrating corresponding staining.
FIgURe 1 | NF-κB in post-mortem tissue from the orbitofrontal cortex
from ASC and control donors. (A) Image of Western blot probed with the
anti-NF-κB p65 and anti-β-actin antibodies (loading control). Molecular mass
markers are shown in kDa. (B) Relative expression of NF-κB p65 subunit,
normalized to the lowest value recorded (Study ID C01). (C) Image of
fractioned samples probed with anti-NF-κB p65. Sample IDs suffixed “/N” are
the nuclear component, and suffixed “/C” the cytosolic component. Sample
IDs prefixed “ A” are from ASC donors, prefixed “C” from control donors.
FIgURe 2 | NF-κB p65 expression in neurons. (A) A representative image of
a neuron stained with anti-β-III Tubulin (green), anti-p65 (red), and DAPI (blue).
Scale bar = 10 μm. (B) Percentage of neurons with anti-p65 nuclear staining
for each sample. Sample IDs prefixed “ A” are from ASC donors, prefixed “C”
from control donors.
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Young et al. Aberrant NF-kappaB expression in ASC
controls. These between-groups differences were increased in
astrocytes and microglia relative to neurons, but particularly
pronounced for highly active microglia identified by anti-CD11c
expressIon of nf-κB p65 In neurons and MIcroglIa: us cohort
Immunoreactivity measurements of samples from the US
cohort stained by anti-Beta III Tubulin for neuronal identifi-
cation revealed that 69% of cells from ASC samples scored 2–3,
whilst 63% of cells from control samples scored 0–1; Table 2.
Nuclear translocation of NF-κB p65 occurred in 20.83 ± 1.72%
of cells in ASC samples compared to 14.60 ± 1.52% controls;
Immunoreactivity analysis of CD11c positive microglia resulted
in 85% of cells from ASC samples scoring 2–3, whilst 53% of cells
from control samples scored 0–1; Table 2. Nuclear translocation
occurred in 90.50 ± 6.66% of cells in ASC samples compared to
22.00 ± 2.00% from controls; Figure 3C.
Due to limited sample volumes, processing and analysis for
astrocytes and CD11b positive microglia was not undertaken.
Differences in immunoreactivity for CD11b positive microglia
were also observed, with 81% of cells from ASC samples scoring
2–3 compared to 59% of cells from control samples scoring 0–1;
Table 2. The between-group difference in nuclear translocation in
these cells was also more pronounced than in neurons. In CD11b
positive microglia, 93.67 ± 3.00% of cells from samples from ASC
patients expressed nuclear NF-κB p65 compared to 64.25 ± 1.26%
from controls; Figure 3B.
Similarly, CD11c positive (highly active, mature) microglia in
samples from ASC donors had raised levels of immunoreactivity
with 88% of cells from ASC samples scoring in the range 2–3 com-
pared to 58% of cells from control samples scoring in the range 0–1;
Table 2. Nuclear NF-κB p65 expression in CD11c positive microglia
from ASC samples was 89.67 ± 2.08% of cells and 34.00 ± 2.16% from
controls; Figure 3C. Furthermore, using 20 visual fields randomly
selected blind to group, the number of active CD11c positive cells
present was 3.75 times greater in tissue from ASC donors; Figure 3E.
In summary, all cell types demonstrated increased extranuclear
and nuclear translocated NF-κB p65 expression in samples of
brain tissue from ASC donors relative to samples from matched
FIgURe 3 | NF-κB p65 expression in astrocytes and microglia. (A)
Percentage of GFAP stained astrocytes with anti-p65 nuclear staining.
(B) Percentage of CD11b stained microglia with anti-p65 nuclear staining. (C)
Percentage of CD11c stained (highly activated) microglia with anti-p65 nuclear
staining. (D) A representative microglial cell with nuclear NF-κB p65 staining
with anti-CD11c (green), anti-p65 (red), and DAPI (blue). Scale bar = 20 μm. (e)
The number of CD11c positive microglia found in 20 fields of random sampling.
Blue bars represent samples from the UK cohort, red bars samples from the US
cohort. Sample IDs prefixed “ A” are from ASC donors, prefixed “C” from
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May 2011 | Volume 2 | Article 27 | 6
Young et al. Aberrant NF-kappaB expression in ASC
ASC (Zimmerman et al., 2005), together with activated astrocyte
and microglia in post-mortem brain tissue (Pardo et al., 2005).
Nevertheless, the underlying molecular events remain unclear.
In this article, for the first time to our knowledge, we report
the aberrant expression of a pro-inflammatory transcription fac-
tor, NF-κB, in samples donated to the London Brain Bank (UK
cohort) and Harvard Brain Tissue Resource Center (US cohort).
This discovery could play a major role in refining diagnostic tests
and therapeutic interventions for ASC. Excess NF-κB p65 expres-
sion was observed in cytosolic, but predominantly nuclear com-
partments in ASC samples (Figure 1). These relative increases were
subsequently localized to neurons, astrocytes, and microglia, but
were particularly pronounced in highly activated (CD11c positive)
microglia. Furthermore, nuclear translocation of NF-κB suggests
activation of the molecule.
NF-κB induces the expression of inflammatory cytokines and
chemokines and, in turn, is induced by them (Barnes and Karin,
1997; Pahl, 1999). This establishes a positive feedback mechanism
(Perkins, 2004), which has the potential, when NF-κB becomes
aberrantly active, to produce the chronic or excessive inflammation
associated with several inflammatory diseases (Barnes and Karin,
1997; Mattson et al., 2000; Mattson and Meffert, 2006; Memet,
Primarily in neurons, NF-κB is activated in order to provide a
protective function. A small, 6% point difference between ASC and
control groups suggests the presence of extensive stress on neurons
These data confirmed results derived from the UK cohort, with
differences in nuclear translocation of NF-κB p65 in neurons 6.23%
points higher for ASC samples from the UK cohort and 4.67% points
higher from the US cohort. In CD11c positive, highly activated
microglia, between-group differences in nuclear translocation of
NF-κB p65 were similarly elevated in the US cohort (68.50% points
higher in ASC samples) and the UK cohort (55.67% points higher).
dIfferences In ph and relatIonshIp to nf-κB p65 expressIon: uK
Measurement of homogenized tissue yielded a 0.92-unit pH between-
group difference (Figure 4A), decreased in ASC samples relative to
control samples from the UK cohort. The relationship between pH and
NF-κB p65 expression was explored by linear regression and a highly
significant effect observed [F(1,5) = 98.3; p = 0.00018; Figure 4B].
Aberrant pH was localized to subcellular compartments by
immunofluorescence. Low pH observed in homogenized tissue
from ASC samples appears to be a result of a reduced pH in the
lysosomal compartments of cells. Tissue from ASC patients had
lysosomes that fluoresced green whereas that from controls fluo-
resced orange; Figures 4C,D.
An emerging focus of research into ASC has suggested neuroin-
flammation as an underlying biological model, with evidence from
irregular cytokine profiles in the cerebrospinal fluid of children with
FIgURe 4 | Measurement of pH. (A) Graph of pH of homogenized tissue
samples. (B) Plot of normalized overall expression of NF-κB p65 from
quantification of Western blot vs. pH of homogenized samples with a
superimposed line of linear regression. Filled circles are samples from ASC
donors, open circles samples from control donors. (C) Immunofluorescent
image from a tissue sample from control donor (×63 objective, scale
bar = 20 μm) and (D) from a sample from ASC donor (×100 objective, scale
bar = 20 μm). Images are pseudocolored and show the ratio of acridine orange/
Lysotracker Red fluorescence. Green hues represent low lysomic pH, and red
hues represent high lysomic pH.
www.frontiersin.org May 2011 | Volume 2 | Article 27 | 7
Young et al. Aberrant NF-kappaB expression in ASC
factor in chronic inflammatory dis-
eases. N. Engl. J. Med. 336, 1066–1071.
Baron-Cohen, S. (2002). The extreme
male brain theory of autism. Trends
Cogn. Sci. 6, 248–254.
Baron-Cohen, S., Leslie, A. M., and Frith,
U. (1985). Does the autistic child have a
“theory of mind”? Cognition 21, 37–46.
Barger, S. W. (2005). Vascular con-
sequences of passive Abeta
immunization for Alzheimer’s
disease. Is avoidance of “malac-
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Barnes, P. J., and Karin, M. (1997). Nuclear
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Anderson, M. P., Hooker, B. S., and
Herbert, M. R. (2008). Bridging from
cells to cognition in autism patho-
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Akiyama, H., Nishimura, T., Kondo, H.,
Ikeda, K., Hayashi, Y., and Mcgeer, P.
L. (1994). Expression of the recep-
tor for macrophage colony stimu-
lating factor by brain microglia
and its upregulation in brains of
patients with Alzheimer’s disease and
Consistent observations of pH reduction in brain tissue from
patients with schizophrenia have unclear origins, with medication
and cause of death effects suggested in addition to it reflecting
features of the disorder (Lipska et al., 2006; Halim et al., 2008).
Thus, there remains the possibility that reductions in pH represent
agonal artifact, and indeed ante mortem hypoxia and long termi-
nal phases as well as gender, are known to lead to pH reductions
in post-mortem brain tissue (Monoranu et al., 2009), although
there is no correlation with PMI and age at death. In this study
donors were matched on all these quantities (Table 1). The linear
modeling between NF-κB concentrations and pH is highly sig-
nificant and furthermore is located in the lysosomes (Figure 4)
Nevertheless, a post-mortem change in pH from chemical cascades
involving NF-κB cannot be excluded. Should further experimen-
tation confirm the relationship between these cellular markers of
inflammation and pH, then this may be a potential biomarker for
diagnosis and response to therapeutic interventions. Measurements
of in vivo intracellular pH can be achieved non-invasively with
phosphorous-31 magnetic resonance spectroscopy (Pettegrew
et al., 1988) or magnetization transfer techniques (Sun et al., 2007).
To summarize: NF-κB is aberrantly expressed in the orbitofrontal
cortex as indicated by measurements on post-mortem tissue from
ASC patients, and particularly in highly activated microglia. This
region is a locus of abnormal function in ASC that underlies the
abnormal development of social and cognitive skills (Sabbagh,
2004). This is the first discovery of its kind that identifies a poten-
tial mechanism for neuroinflammation in ASC through increased
expression of this pro-inflammatory molecule and the significant
involvement of resident immune cells. The connection of this result
to changes in intracellular acidity indicates an investigation of pH
across the entire brain parenchyma in living patients.
Whilst evidence of causal link remains to be established, the idea
that the induction of inflammation via the NF-κB signaling cascade
is observed in regions of the neocortex associated with behavioral
and clinical symptoms of ASC gives credence and impetus to inter-
ventions focusing on this potential therapeutic target.
This work was supported by grants to Adam Young from Archimedes
Pharmaceuticals, the University of St Andrews, and the Pathological
Society of Great Britain and Ireland. We are grateful to J. Pickett
and C. Eberhart at the Autism Tissue Program and the Harvard
University, and C. Troakes at King’s College London Brain Bank,
Institute of Psychiatry for providing samples. We thank Dr G.
Cramb for his helpful comments and assistance. Finally, we extend
our gratitude to the families of the donors for supporting the tissue
in ASC is unlikely. For confirmation, the cell morphology of neurons
was screened (Mpoke and Wolfe, 2003) for signs of apoptosis or
necrosis to assess the relative rates of cell death. There were minimal,
if any, differences between-groups, an observation that concurs with
work by Hausmann et al. (2004) who reported that apoptosis was not
detected in non-traumatically injured brain tissue when the PMI was
less than 72 h. The samples reported in the study fall into this category
(Table 1). Although needing to be confirmed at the molecular level,
this may well be a key finding as it demonstrates the potential revers-
ibility of the condition, something not commonly observed in many
neurological disorders where there is high irreversible cell death.
The elevated nuclear translocation in ASC samples (Figure 3)
supports previous work on astrocyte and microglia activation in the
condition (Pardo et al., 2005; Vargas et al., 2005; Zimmerman et al.,
2005; Anderson et al., 2008). The activation of microglia induces an
array of cellular events which accumulate to reduce neural function.
This is potentially of interest more widely as previous studies have
identified a potential link between low pH of homogenized tissue
and learning disabilities (Rae et al., 2003) as well as Alzheimer’s
disease (Majumdar et al., 2007).
Confirmation of the immunofluorescence results was obtained
from an independent set of samples from the Autism Tissue Program
at the Harvard Brain Tissue Resource Center (US cohort). Close
correspondence in magnitude and direction of between-group dif-
ferences with the UK cohort was observed. However, it is worthy of
note that samples from the US cohort were donated by people very
much younger than the UK cohort. Thus, as well as validating the
results from the UK cohort, the observation of aberrant expression
of NF-κB can be extended to cover an age range from 5 to 40 years.
While the origin of inflammatory signaling in ASC remains
undetermined, genetic or epigenetic factors are mechanisms
which can subsequently up-regulate the NF-κB signaling cascade.
Animals subject to prenatal immunological challenges during early
gestation subsequently displayed marked learning deficits (Meyer
et al., 2006) and morphological brain changes post-natally (Li et al.,
2009). Extracellular detection of pathogens by toll-like receptors
leads to signaling pathways resulting in over-expression of NF-κB.
Theoretically, this would allow for the range of environmental
stimuli which are associated with the condition to act on a central
node of the inflammatory component of the condition. Supported
by the increase in NF-κB expression at the protein level, an inher-
ited component is most likely why the chronic inflammatory state
maintains throughout adulthood.
The ∼1 unit pH difference observed in homogenate brain tissue
from controls and ASC patients (Figure 4A) appears to be a result of
increased lysosomal activity (Figures 4C,D). Coupled to the highly
significant linear relationship between pH and NF-κB (Figure 4B),
the inference is that acidification does not influence cognitive func-
tion directly, but is a consequence of neuroinflammation.
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Conflict of Interest Statement: The
authors declare that the research was con-
ducted in the absence of any commercial
or financial relationships that could be
construed as a potential conflict of interest.
Received: 10 December 2010; accepted: 03
May 2011; published online: 13 May 2011.
Citation: Young AMH, Campbell E, Lynch
S, Suckling J and Powis SJ (2011) Aberrant
NF-kappaB expression in autism spectrum
condition: a mechanism for neuroinflam-
mation. Front. Psychiatry 2:27. doi:
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