Molecular Neuroscience
Head: Dr. Hubert H.M. Van Tol
The goal of the Molecular Neuroscience Section is to understand in detail
the mechanisms by which neural communication takes place. By taking a
deterministic approach to fundamental problems in neurotransmission, we
seek to understand the components involved in communication between neurons
and how these molecules may contribute to mental illness as well as how
they serve as therapeutic target.
The section currently has three principal investigators directing their
own research groups. Their research involve molecular, biochemical and
electrophysiological approaches to studying the molecules involved in
neuronal signalling. The scientists in the section principally apply in
vitro approaches and use of model systems, including transgenic mice and
the nematode C.elegans, for their research. They often extend their
findings to human disease by collaborating with other scientists, most
notably the Neurogenetics Section at CAMH. The research section is widely
associated with many neuroscientists within and outside Toronto, and is
associated with the CIHR groups Dopamine and Psychomotor Disease; and
The Synapse (http://www.utoronto.ca/synapse/).
Molecular
Neurobiology I
Dr. Hubert H.M. Van Tol
This group focuses on the dopamine signalling system in the central nervous
system. This system is often presumed to be the origin, and/or one of
the main targets for therapeutic intervention, for the symptoms of several
psychiatric and neurological disorders, including schizophrenia, bipolar
disorder, Huntington's disease, Parkinson's disease, Tourette's syndrome,
addictions and attention deficit hyperactivity disorder. To date, five
different dopamine receptors, members of the G protein-coupled receptor
(GPCR) family, have been identified in humans. We wish to obtain a complete
understanding of the individual components of the dopamine signalling
system, so we can evaluate the contribution of the system to development
of disease, improve therapeutic intervention and minimize treatment side-effects.
In humans, the neurotransmitter dopamine is synthesized in the brain
in neurons located in the mid-brain area, most notably the substantia
nigra and the ventral tegmental area. These neurons project to their target
areas, where dopamine is released in a regulated manner.
The importance of proper function of these neurons is seen in examples
such as the loss of dopamine neurons of the substantia nigra, which is
the cause for Parkinson's disease. In schizophrenia research, evidence
is emerging for an excess of dopamine release, and in addictions, several
drugs of abuse stimulate dopaminergic transmission.
Dopamine released from neurons will bind to specific targets known as
dopamine receptors. Dopamine receptors are not only on the postsynaptic
neurons, but they are also present on the dopamine-synthesizing presynaptic
neurons in the brain. Thus dopamine receptors can serve as a component
of the feedback mechanism for controlling release. Activation of the dopamine
receptors by the neurotransmitter activates a cascade of intracellular
signalling molecules. This cascade will ultimately mediate a change in
the activity of various ion channels or modulate the status or expression
of the molecules involved in neurotransmission, thus modulating the excitability
of the cell and the transmission of a signal. Many elements of the system
are still poorly understood, such as the factors controlling dopamine
neuron development, regulation of neurotransmitter release, and mechanisms
of dopamine-receptor mediated changes in intracellular signalling.
Novel Dopamine Signalling
Pathways
Dopamine receptors belong to the superfamily of receptors that mediate
their signal through heterotrimeric G proteins. In the last few years,
evidence has been emerging that this family of receptors may also directly
interact with other cellular components that will either regulate the
receptor or serve as effector. Using large-scale yeast two-hybrid and
phage displays screening protocols, and more targeted screens, we have
found that dopamine receptors can bind Src homology 3 (SH3) domains. SH3
domains can be found in a variety of proteins involved in intracellular
signalling, and these SH3 domains play a role in bringing proteins together
in the cell. We have observed that this type of interaction may modulate
receptor-mediated activation of the mitogen-activated protein kinases
(MAPK) and receptor internalization.
We recently found that dopamine D2 and D4 receptors activate the MAPK
pathway through the process of transactivation, by which platelet-derived
growth factor receptors are activated. In collaboration with Dr. J.F.
MacDonald (Department of Physiology, University of Toronto) we have found
that transactivation is also critical for the mechanism by which dopamine
receptors can reduce N-methyl-D-aspartate (NMDA) type glutamate receptor
activation in hippocampal neurons. The mechanism of transactivation is
not very well understood and is the further subject of our current studies.
The observation that dopamine receptors can transactivate growth factor
receptors, and thus a large variety of intracellular signalling pathways,
may give us new insight into how dopamine receptors control neuronal development,
survival, differentiation and synaptic plasticity.
GIRK Channel Complex
G protein-activated inwardly rectifying K+ channels (GIRK; a.k.a. Kir3)
are the effector of various G protein coupled receptors, including the
dopamine D2, D3 and D4 receptors. Four different Kir channel subunits
have been identified, namely Kir3.1, 3.2, 3.3 and 3.4. These channel subunits
form a tetrameric complex to make a functional channel. The physiological
importance of these channels lies in their ability to maintain the membrane
potential to the resting potential, and thus regulate the excitability
of the cell. In this regard, the presence of these channels in the presynaptic
dopamine neurons, particularly Kir3.2, may play an important role in the
feedback regulation of dopamine release through its activation via presynaptic
dopamine D2 receptors.
We know these channels are activated in a membrane-delimited manner,
arguing that the channel and receptor have to be in close proximity of
each other to mediate functional activation. However, we do not know the
precise nature of the channel/receptor relationship. By using molecular
and biochemical approaches, we have now established that the dopamine
receptor and GIRK channels form a stable complex early during their synthesis.
The stability of the receptor-channel complex is not dependent on receptor
activation or G proteins, but its initial formation is dependent on G
beta gamma protein subunits. The observation that the receptor-channel
complex is stable may have important implications on how temporal control
of the synthesis of the individual components regulates the activation
of different signalling pathways by GPCRs. We are now further investigating
the molecular determinants of this interaction.
Model systems: C.elegans
and Dopamine Signalling
The nematode C.elegans is a model system amenable to analysis
with powerful genetic tools. Its genetics, anatomy, development, behaviour
and nervous system have been well studied. Although C.elegans,
by mammalian standards, has a very simple nervous system, analysis of
the genomic sequence suggests it contains genes encoding most of the known
molecular components of mammalian brains. Various lines of genetic and
biochemical evidence suggest that C.elegans has a dopamine system.
Using high performance liquid chromatography techniques coupled with
electrochemical detection, we have confirmed that C.elegans indeed
produces dopamine and several of dopamine's metabolic products. Furthermore,
using bioinformatics combined with genetic and molecular approaches, others
have identified several key components of its dopamine system, including
tyrosine hydroxylase and the dopamine transporter.
The dopamine receptor of C.elegans has thus far remained elusive.
Using bioinformatic approaches, we have identified up to 15 candidate
dopamine receptors. One of these receptors encodes on functional and pharmacological
grounds for a dopaminergic receptor. In collaboration with Dr. J. Culotti
(Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto),
we identified the neurons in which this receptor is expressed in C.elegans
and mutant strains in which the receptor is disabled. These mutant
strains are now being analyzed to establish the functional role of the
receptor, so we next can employ genetic suppressor screens to identify
genes linked to the dopamine receptor functioning. This way, we hope to
identify new components involved in functioning of the dopamine signalling
system. Based on the observed genetic similarities between humans and
C.elegans, these genes may fulfill a similar role in the functioning
of the mammalian dopamine system.
Model Systems: Candidate
Genes for Schizophrenia Using a
Rodent Model System
Schizophrenia is thought to be a complex genetic disorder best reflected
by a multiplicative multilocus model. This complexity forms a huge challenge
for genetic studies, a challenge best met by the use of a candidate gene
analysis in family-based association studies. The selection of candidate
genes for these studies is thus far predominantly based on the role of
such genes in development, the functioning of the dopamine system, or
on these genes being targeted for drugs inducing or treating psychosis.
Various new molecular technologies, particularly micro-array technologies,
allow for the rapid screen of the expression of many genes. Genes with
an altered expression in schizophrenia may therefore be labelled as candidate
disease genes.
In collaboration with Drs. Lipska and Weinberger (Clinical Brain Disorders
Branch, NIMH), we pursued the use of a non-human model system for schizophrenia
in a screen for candidate genes. This model, developed by Lipska et al.
(1993), contains not only the appropriate behavioural abnormalities, but
also the delayed developmental component and differences in genetic susceptibility
for the disorder. To date, we have screened up to 30,000 genes for six
different parameters of the model, and have identified several genes that
may be involved in the disorder. The selection of candidate genes is essentially
a cross-section of genes with altered expression under the different model
parameters. Several of the identified genes are now being analyzed in
genetic family-based association studies in collaboration with Drs. J.L.
Kennedy and F. Macciardi (Neurogenetics
Section).
Molecular
Neurobiology II
Dr. Fang Lui
Neurotransmitters, such as dopamine, GABA and NMDA, are chemicals in
the central nervous system (CNS) that exert their physiological function
by binding to receptor proteins. These proteins allow neurotransmitters
to communicate and send important information between neurons. The major
focus in our lab has been the identification of interactions between dopamine
receptor/transporters with other modifying accessory proteins in the CNS.
These interactions have physiological implications for both normal and
disease states.
Our ongoing research projects include the following.
Functional Cross-Talk
between Dopamine D1 Receptor and
NMDA Receptors
Numerous studies have demonstrated the functional interaction between
dopamine D1-like receptors and ligand-gated ion channels, such as NMDA
and GABAA receptors. However, the mechanism through which these two receptor
families interact remains unclear. The subcellular distribution patterns
of these receptor/ion channels have suggested that D1 and D5 receptors
may preferentially modulate excitatory and inhibitory inputs through the
interactions with the NMDA or GABAA receptors respectively. We found that
dopamine D5 receptors can regulate inhibitory neurotransmission through
direct protein-protein coupling with GABAA receptors (Liu et al., 2000).
Therefore, we examined whether dopamine regulation of excitatory neurotransmission
can be mediated by a functional interaction of dopamine D1 and NMDA receptors
through direct protein-protein coupling. To date, we have found that dopamine
D1 receptors can modulate both the excitatory neurotransmission and excitotoxicity
mediated by the NMDA glutamate receptor through two distinct pathways
formed by direct protein-protein binding. We are currently investigating
the mechanisms by which dopamine receptors modulate NMDA receptor mediated
excitotoxicity. We are also investigating whether NMDA receptors can modulate
dopamine receptor function through direct protein-protein interaction.
A manuscript based partially on this work has been submitted for publication.
Regulation of Dopamine
Transporter Function by Dopamine D2 Receptor
The primary mechanism for the inactivation/recycling of dopamine released
into the synaptic cleft is the reuptake of dopamine into the presynaptic
dopaminergic neuron via the dopamine transporter (DAT). This ability of
the DAT to control synaptic dopamine levels suggests the importance of
DAT regulation.
DAT regulation may be mediated by second messengers and their effectors
(e.g., arachidonic acid, Ca2+ levels,
calmodulin-dependent kinases, and PKA- and PKC-dependent pathways). Nonetheless,
the most important method of DAT regulation may be initiated by presynaptic
D2 receptors.
The activation of D2 receptors in regulating DA reuptake has been implicated
in previous studies. We have shown that DAT activity is upregulated in
cells co-expressing both recombinant DAT and D2 receptors. However, as
opposed to previous studies, this reuptake was independent of D2 activation
by agonist pretreatment, a finding that is supported by the inability
of the presence of D2 antagonists to attenuate this upregulation. Interestingly,
confocal immunofluorescence of cells co-expressing both DAT and D2 receptors
show a robust translocation of DAT protein from the intracellular region
to the cell surface, independent of D2 agonist/antagonist treatment.
These studies suggest that D2 regulation of DAT may involve either direct
or indirect interactions by mechanisms involved in the trafficking of
DAT protein to the cell surface and/or through interactions with G-proteins,
associated with D2 receptors.
Molecular
Physiology
Dr. Xian-Min Yu
The pathophysiological process underlying the development of schizophrenia
remains a mystery for modern medicine. Since the discovery that antagonizing
the NMDA type glutamate receptor may induce schizophrenia-like symptoms,
more and more data obtained from clinical and basic research studies have
convincingly indicated that abnormal NMDA receptor activity is an important
factor in the development of schizophrenia.
The long-term goal of our research is to characterize mechanisms underlying
the activity-dependent neuroplasticity associated with physiological and
pathological processes in the central nervous system (CNS). To answer
the fundamental question of how neuronal activity can alter synaptic responses
in the CNS, we have focused on identifying novel mediator(s) that may
couple the neuronal activity to the modulation of neurotransmitter functions
through intracellular mechanisms. Previously, we have demonstrated that
Na+ is such a mediator for NMDA receptor up-regulation and that the Na+
action on NMDA receptors is regulated by NMDA receptor-associated Src-family
protein tyrosine kinases. Based on these findings, we have launched further
studies focusing on the organization of NMDA receptor-associated signalling
complex and on the role of Na+ in the coupling of extracellular events,
such as lowering extracellular Ca2+
concentration, to the modulation of NMDA receptor activity.
In a collaborative project with Dr. F. Liu's group, we are investigating
the regulation of GABAa and NMDA channels by a novel protein-protein binding
interaction between dopamine D1 receptors and these ligand-gated ion channels,
originally identified by Drs. H.B. Niznik and F. Liu. Findings in this
project demonstrate a new theory that different types of neurotransmitter
receptors may directly modulate each other via their direct binding interaction
(Nature, 2000, 403, 274-280).
Our findings have laid the basis for a novel concept in the regulation
of NMDA receptor function in the CNS, which may prove important for developing
novel therapeutic approaches to treat a wide range of clinical problems,
including schizophrenia.
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