Autism is a complex disorder of the brain that lasts throughout a person’s life and affects the ability to communicate, form relationships with others, and respond appropriately to the environment typically by 3 years of age. In the past two decades autism became a primarily disorder of brain development. The incidence of Autism Spectrum Disorders is currently 1 in 150 and it affects boys four times more often than girls. As many as 20 or more genes on different chromosomes may be involved in the disorder to various degrees and determine the severity of symptoms. Interactions between those genes in addition to environmental factors likely contribute to variable expression of autism-related traits. The multidisciplinary research program in my laboratory integrates genetics, molecular and cellular neurobiology approaches to study the link between the causative biological factors and behaviour.
Current Research Studies in my Lab:
STUDY 1: Characterization of Genetic Risk Factors
Associated with Autism Spectrum Disorders
Early brain development relies largely on intrinsic pattern of gene expression and may be influenced by genetic and/or environmental predispositions. Therefore, early neuronal development can be vulnerable to gene dosage changes. In this study we measure the differences in gene expression level in affected individuals compared to normally developing children to assess what might be different in the brain of a child with autism. We also search for common heritable markers in siblings, twins and parents. This study enables identification of genetic causes and will reveal common metabolic and signaling pathways that might be defected in the nervous system or causing other physiological symptoms found in autism such as gastro-intestinal, immune, motor, or anxiety.
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Gene
expression profiling is used in
our lab for detection of genes affected in Autism Spectrum
Disorders. It provides a
global view of functional gene networks that might be altered in the
brain, it allows identification of compromised pathways and provides a
meaningful approach towards understanding the complex biology of autism
disorders. Our goal is to further our understanding of molecular
changes
occurring in the brain and how they might contribute to various symptoms. Gene
expression analysis involves fewer steps, gives
the possibility to evaluate a wide range of candidate genes and can be
a useful
tool for diagnosis of autism earlier
in life before the symptoms become noticeable. We
collect mouth epithelial cells (buccal
cells) using a gentle brush. It is a non-invasive method especially
useful for
very young subjects.
We
are currently seeking participants of all ages, siblings, twins, parents
and other family members for our study. |
STUDY 2: Environmental Factor Contributing to
Autism Spectrum Disorders
The plasma membrane phospholipids play an important role in the nervous system and serve as a supply of second messenger molecules important for normal functioning of the brain. The emerging evidence implicates that abnormal fatty acid metabolism may play a contributing role in the pathology of autism. Fatty acids homeostasis may be altered in autism as a result of insufficient dietary supplementation, genetic defects, function of enzymes involved in their metabolism, or influence of various environmental agents such as drugs such as misoprostol, infections or inflammation.
The plasma membrane phospholipids play an important role in the nervous system and serve as a supply of second messenger molecules important for normal functioning of the brain. The emerging evidence implicates that abnormal fatty acid metabolism may play a contributing role in the pathology of autism. Fatty acids homeostasis may be altered in autism as a result of insufficient dietary supplementation, genetic defects, function of enzymes involved in their metabolism, or influence of various environmental agents such as drugs such as misoprostol, infections or inflammation.
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Neurobiology of Lipid Signaling Various
bioactive fatty acid
molecules such as arachidonic acid (AA) can
be
normally released from membrane phospholipids by the action of phospholipase
A2 (PLA2)
and
subsequently metabolized into various types of bioactive prostanoids.
Cyclooxygenase-1 enzyme (COX-1), constitutive form, or cyclooxygenase-2
(COX-2), inducible form, converts AA to the unstable PGG2
intermediate and then to the prostanoid precursor PGH2,
which is
further metabolized by the prostaglandin (PG) synthase into the major lipid signaling messengers
such as prostaglandins (PGE2). PGE2 is an important signaling molecule involved
in inflammation, pain or synaptic plasticity in the
nervous system. It exerts its effects
through activation of their respective G-protein-coupled receptors (GPCRs) called
EP
receptors (EP1-4). Our
lab investigates how
alterations in
this pathway affect cell function and contribute to brain dysfunctions
seen in autism. We use
various cell cultures (neuronal stem cells, neurons and astrocytes) and
mouse as experimental model systems.
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Prostaglandin E2 and misoprostol induce neurite retraction in Neuro-2a cells Prostaglandin
E2 (PGE2)
is a key lipid-derived compound which mediates important physiological
functions in the nervous system Recent studies have shown that elevated
levels of lipid peroxidation
and
oxidative stress biomarkers and prostaglandin metabolites may underlie
some pathologies of the
nervous
system. The prenatal exposure
to a drug misoprostol, a prostaglandin type E analogue, has also been
linked to
a number of neurodevelopmental defects. Using ratiometric calcium
imaging with
fura-2AM (left) as a calcium
indicator (at 340nm when bound to calcium) we
test the dose-dependent effects of PGE2
and misoprostol on calcium homeostasis in neuronal type cells
(Neuro-2a), astrocytes type I and neuronal stem cells. Our recent
results show that both drugs increase the
amplitude of
calcium transients in growth cone of Neuro-2a cells and induce neurite
retraction (below). This might
have significant implications for neuronal
differentiation and cell comunication in the nervous system.
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Misoprostol elevates intracellular calcium in Neuro-2a cells Studies in
our lab have also provided
evidence for the involvement of prostaglandin E2 (PGE2)
signaling in abnormal intracellular calcium homeostasis and its
potential affect on
the
nervous system. Misoprostol, a prostaglandin type E analogue, has been
implicated in a number of neurodevelopmental disorders. Our lab
investigates
its mode of action in the nervous system. Misoprostol acts on the same
receptors as PGE2, a natural
lipid-derived compound, which mediates important physiological
functions in the
nervous system via activation of the EP receptors (EP1-4). Using
ratiometric
calcium imaging with fura-2 AM we
have shown
that misoprostol alters intracellular calcium levels in mouse
neuroblastoma
(Neuro-2a) cells via similar mechanisms as PGE2. We have
demonstrated
in that the misoprostol-induced increase in calcium is mediated by a
protein
kinase A (PKA)-dependent mechanism and that the EP4 receptor signalling
pathway
may play an inhibitory role on calcium regulation.
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Expression pattern of EP receptors during mouse embryonic development Quantitative
real-time PCR revealed that the
mRNA
expression level of the four EP receptors was significantly higher
during the
neurogenesis period in mouse indicating the importance of PGE2
signalling in early neuronal development. We assessed
the expression
level of mRNA for the EP receptors (EP1, EP2, EP3α, EP3β,
EP3γ and EP4) in
mouse embryos at days 7, 11, 15 and 17
(E7, E11, E15 and E17). The relative quantification (RQ) of EP1, EP2, EP3α and EP3β
receptors exhibits a significant
increase in the expression level at E15 (peak of neurogenesis; day
12-17). EP3α and EP3β
expression level was also
significantly higher at E7, part of organogenesis (prior to day 12). The EP3γ receptor
shows relatively uniform expression in all
developmental stages with E7 being the highest expression. The EP4
receptor was
predominantly expressed at significantly higher levels (about 150-fold
increase) at E7 as compared to the adult brain. The results indicate
that EP receptors
may play an imortant role during the critical period and that changes
in the
level of
endogenous PGE2 or exogenous drug misoprostol may have
profound
effects on the developing nervous system.
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Most recent publications:
Tamiji J. and Crawford D.A. The Neurobiology of
Lipid Metabolism in Autism Spectrum Disorders. NeuroSignals.
2010;18(2):98-112.
pdf
Tamiji J. and Crawford D.A. Misoprostol-induced
elevation of intracellular calcium in Neuro2a cells requires protein
kinase A. Biochemical and Biophysical Research
Communication. 2010. Sept 3;399(4):565-70. pdf
Tamiji J. and Crawford D.A. Prostaglandin E2 and misoprostol induce neurite retraction in Neuro-2a cells. Biochemical and Biophysical Research Communication. 2010. Jul 30;398(3):450-456. pdf
Vallipuram
J., Grenville J.
and Crawford D.A. The E646D-ATP13A4 mutation associated with
autism
reveals a defect in calcium regulation. Cellular and Molecular
Neurobiology.
2010. Mar;30(2):233-46. pdf
Kwasnicka-Crawford
D.A.,
Roberts W., Li M., and Scherer S.W. Characterization of an
autism-associated
segmental maternal heterodisomy of the chromosome 15q11-13 region. Journal
of Autism Developmental Disorders. 2007 Apr;37(4):694-702. pdf
Kwasnicka-Crawford
DA,
Kwasnicka-Crawford
D.A.,
and Vincent S.R. Role of a novel dual flavin reductase
Funding:
The Alva Foundation
National Sciences and Engineering Research Council of Canada (NSERC)
Canada Foundation for Innovation (CFI)
Ontario
Faculty of Health, York University
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