Neuralized expression in third larval instar wing imaginal disc. Picture from Lara Skwarek.
·
Neuronal
Development
· Regulation of Synaptic Transmission, Learning and Memory
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Drosophila Models of Aging and Neurodegenerative Diseases
MAJOR
RESEARCH ACTIVITIES
My
laboratory has been using powerful genetic techniques available in Drosophila,
to study the development and the function of the nervous system.
More recently, we have exploited the knowledge and tools obtained during
the course of these studies to develop models of aging and human
neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) and
Alzheimer’s Disease (AD). A brief overview of each of these areas is described
below.
Drosophila
Models of Neural Development
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Normal
development requires the commitment of individual cells to the appropriate cell
fate and their subsequent differentiation. The long-term goal of this research
is to understand how individual cell fates are acquired during neural
development. To this end, we have
studied the Notch signaling pathway that has been shown to determine the fate of
virtually every cell type in many complex organisms.
Specifically, we have characterized a novel component of this pathway, neuralized, which we cloned several years ago.
More recently, we have shown that neuralized
is required to specify the fate of neural versus epidermal cells during the
development of the adult peripheral nervous system.
Furthermore, using a combination of in
vitro and in vivo assays we, and
others, have shown that Neuralized functions as an E3-ubiquitin ligase that
targets the Notch ligand, Delta, for ubiquitination and subsequent
internalization within cells. These
studies provided the first evidence that Notch signaling could be regulated by
ubiquitination and trafficking of its ligands.
Current efforts are aimed at understanding the precise mechanisms by
which Neuralized regulates Delta trafficking within the cell and how this
regulates Notch signaling.
In
addition to our work on neuralized, we
have also studied the function of presenilins which were first identified as
causative factors in AD but have since been shown to play a role in the Notch
pathway during development. We found that presenilin is required for processing
and trafficking of the Notch receptor during development. Our current efforts
(see below) are aimed at identifying other proteins that can interact with
presenilin and modify its function. These
studies will reveal the role of presenilin in normal development and may also
shed light into its pathological roles in AD.
Drosophila
Models of Neural Function
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Fast
and efficient synaptic transmission depends on mechanisms that regulate both the
release (exocytosis) of synaptic vesicles at the nerve terminal and their
recycling (endocytosis). My lab has
focused on understanding the mechanisms that regulate both processes in vivo.
To this end, we have used a multidisciplinary approach that combines
genetics, molecular and cellular biology, biochemistry, electron microscopy and
electrophysiology to identify and study the function of specific molecules
required for both exocytosis and endocytosis in Drosophila.
Using molecular and biochemical approaches, we were able to identify
novel proteins and determine their role in synaptic vesicle exocytosis.
We have also shown that many of these molecules can regulate additional
membrane trafficking events within the cell and are required for Notch-dependent
signaling during development. Importantly,
we have shown that regulating the levels of specific proteins, known as SNARES,
alters the ability of neurons to sense calcium, which is essential for
neurotransmitter release and all brain function including learning and memory.
During the course of these studies, we developed efficient methods of
measuring synaptic transmission using electrophysiological approaches and also
methods to measure defects in learning and memory in flies.
We are currently using
these approaches to characterize novel mutants that affect learning and memory
in the fly and also to analyze defects in our fly models of neurodegenerative
disease.
Drosophila
Models of Aging and
Human Neurodegenerative Diseases.
Several genes have been identified as causative or risk factors in neurodegenerative diseases such as ALS and AD.
However,
these genes only account for a small percentage of all ALS or AD cases.
Similarly,
although there is increasing evidence that specific genes can affect aging the
identity of these
genes remains largely unknown. We have taken advantage of powerful approaches
available in Drosophila to study aging and human neurodegenerative diseases.
Initially, we developed a model to study ALS and the role of mutations in
the Cu/Zn SOD gene on neuronal survival. In collaboration with Drs. J. Phillips
and A. Hilliker at the U.Guelph, we showed that mutations in Drosophila SOD
result in significant neural loss and premature death, phenotypes similar in
principle to the effects of SOD mutations in ALS. This was the first evidence
that such a simple
organism could be used as a model for complex human diseases. We have also
investigated the effect of mutations in SOD associated with familial ALS in the
motorneurons of transgenic flies to gain insight into the
mechanisms leading to motorneuron cell death. We are currently using electro-
physiological techniques developed during the course of our studies on SV
exocytosis and endocytosis to analyze the phenotypes of both SOD mutants and
transgenics. During the course of
these studies, we also found that overexpression of human SOD in the
motorneurons of wildtype flies could extend lifespan
by
up to 40%. This exciting discovery shows that lifespan can be genetically
controlled and that specific cell
types, including the motorneuron, may be particularly vulnerable to the effects
of aging. We are currently using powerful genetic approaches available in
Drosophila to identify cell types that are susceptible to the effects of aging
and, more importantly, to find new genes that can lengthen the lifespan of an
organism.
We
have also used Drosophila to develop a model of AD. To this end, we have cloned
and molecularly characterized the presenilin gene from Drosophila
(psn). Presenilins (PS), are members of a family of multipass transmembrane
domain proteins that were initially identified as causative factors in familial
AD. Since then, PSs have also been shown to play a role during normal
development through their actions within the Notch signaling pathway, which we
have extensively studied. To determine the function of psn, we have generated
both mutants and transgenics that express either wildtype or FAD-linked
mutations. Using these tools, we found that psn is required for neuronal
differentiation and that it affects Notch subcellular localization and
signaling. We are currently using the genetic and biochemical techniques
developed in my lab to study neuronal development to gain insight into the
precise role of psn in Notch signaling and neurogenesis.
More recently, we have also shown that loss of function mutations in psn
give rise to defects in synaptic plasticity and learning.
We are currently using a combination of electrophysiological and
pharmacological approaches to determine the precise nature of these defects and
whether these can provide insight into the cognitive and memory defects
associated with AD. Finally, we are
also searching for additional genes that may be causative or risk factors in AD.
To this end, we have identified several genetic modifiers of presenilins,
and these are currently being studied in both flies and mice where their role in
AD can be rigorously tested. The identification of these genes will provide
insight into the normal function of PSs and they themselves could represent
causative or risk factors in AD, providing additional therapeutic targets for
this devastating disease.
INTELLECTUAL PROPERTY/LICENSING OPPORTUNITIES
Presenilin Modifiers
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Hospital for Sick Children |
