April 07, 2005
Controlling Brain Wiring With the Flick of a Chemical Switch
With the flick of a chemical switch, researchers can now exert
unprecedented control over the activity of molecules that help wire the
developing brains of mice.
The new technique permits researchers to use drugs to switch the
molecules on and off as precisely and reversibly as a light switch
controls a lamp. Current genetic and chemical manipulation techniques
are more akin to eliminating entire electrical circuits or breaking the
light bulbs in the lamps.
“I am so impressed with how well these in vivo experiments have gone that there is a good argument that a large number of protein kinases should be targeted in this way.”
David D. Ginty
The researchers said the technique will enable them to explore how
the molecules, which are called neurotrophins, regulate the growth and
survival of neurons in newborn and adult animals. Studying the
regulation of neurotrophins is important because it will help
researchers understand how the brain develops and functions in both
normal and diseased states, they said. For example, neurotrophins play
a role in supporting survival of neurons that are lost in
neurodegenerative diseases such as Alzheimer's disease.
The researchers described their chemical-genetic approach to
controlling neurotrophin signaling in the April 7, 2005, issue of the
journal Neuron. They were led by Howard Hughes Medical Institute
investigator David D. Ginty at the Johns Hopkins University School of
Medicine, and Pamela England and Kevan Shokat at the University of
California, San Francisco. Shokat was among the 43 scientists recently
selected in a nationwide competition to become HHMI investigators.
In their studies, the scientists sought to control neuronal growth
more precisely by employing nerve growth factor (NGF) and brain-derived
neurotrophic factor (BDNF). These neurotrophins regulate neuronal
growth by activating specific tyrosine kinase (Trk) receptors on the
surface of neurons. The Trk receptors translate the neurotrophin
signals to regulate neuronal growth and survival machinery.
Traditionally, when researchers wanted to study neurotrophin
function in mice, they either knocked out those genes completely or
attempted to switch them off after birth using genetic manipulation,
drugs, or antibodies. According to Ginty, all these techniques have
significant drawbacks.
“All the neurotrophin knockouts are lethal around
birth,” said Ginty. “And other techniques of conditionally
knocking out the genes after birth are limited in their application and
are irreversible.” Drugs and antibodies that target neurotrophins
are not specific enough, Ginty said. Antibodies are further limited
because they trigger a general immune response and cannot cross the
blood-brain barrier to infuse into brain tissues.
Shokat and his colleagues, however, had developed a technique for
mutating a single amino acid in protein kinases that rendered these
enzymes susceptible to kinase-inhibiting drugs that target only a
specific type of kinase. The mutations have no other effect on the
kinases' functions.
Ginty and his colleagues applied the chemical-genetic technique,
which had been developed in cultured cells, to animals. They found that
they could specifically deactivate the function of mutated receptors
for either NGF, BDNF or NT-3 with the Trk-inhibiting drugs. When the
drug was removed, the receptors would reactivate to their normal
function. Thus, said Ginty, the new approach represents a powerful
technique for exploring the neurotrophins by switching them off and on
at will.
“A major advantage of this approach is that one has an ideal
experimental control animal in the wild-type mouse, which has a normal
amino acid instead of the mutation at the key position,” said
Ginty. “Such an ideal control animal is something that one
rarely, if ever, has in such experiments. Also critical is that since
this is a pharmacological approach to controlling neurotrophin activity
it is rapidly acting and reversible.”
Such a chemical-genetic approach in mice should be widely
applicable, given that cells use a vast array of kinase switches, Ginty
said. “I am so impressed with how well these in vivo experiments
have gone that there is a good argument that a large number of protein
kinases should be targeted in this way. There are over five hundred
protein kinases in the genome and we have good inhibitors for only a
very small number of them.”
The chemical-genetic approach could offer a far more comprehensive
picture of neurotrophin function, said Ginty. “The null mutations
reveal a phenotype, but if it's a lethal phenotype then you never
understand the full range of functions of that molecule,” said
Ginty. “Often times in the animal with a null mutation, one only
sees the first function, and so you miss all the others,” he
said.
Ginty said that he and his colleagues plan to use the technique to
explore how neurotrophins and their receptors control development of
the forebrain immediately after birth. “A lot of the
developmental events that control patterning of the forebrain occur
postnatally, and until this technique, the roles of neurotrophins have
been very difficult to study,” he said. In addition, the
technique can be used to explore where in the extensive geography of
the neuron a particular neurotrophin receptor functions to regulate a
particular developmental event, said Ginty.
“I am also hoping that other laboratories will use these
engineered mice to study the role of neurotrophins in adult animals —
for example in learning, memory, and neural plasticity. With this
technique, people can ask questions about windows of time during which
these molecules contribute to a given function, because it's possible
to inhibit with fine temporal control and reversiblity,” he
said.
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