May 25, 2001
Visualizing Protein Synthesis in Living Neurons
Over the last two decades, neuroscientists have been
building the argument, buttressed by data from many experiments, that dendrites
— the fine fibers that extend from neurons — can synthesize proteins. If
dendrites can synthesize proteins, they may also have the capacity to modulate
the strength of connections between neurons and ultimately influence neural
activities, including learning and memory.
In a research article
published in the May 2001 issue of the journal
Neuron,
Howard Hughes Medical Institute investigator
Erin M. Schuman
and colleagues at the California Institute of Technology describe a new
technique that has allowed them to show that protein synthesis occurs in intact
dendrites. The technique relies on a reporter molecule constructed by Schuman
and colleagues, Girish Aakalu, Bryan Smith, Nhien Nguyen and Changan Jiang.
When the molecule is introduced into neurons, it emits a telltale glow if
protein synthesis is occurring.
The images produced by
Schuman’s team demonstrate that protein synthesis takes place locally in the
dendrites. “There was early evidence that protein-synthesis machinery was
present in dendrites,” said Schuman. “Those findings were intriguing because
they implied that dendrites had the capacity to make their own proteins.”
The idea that dendrites
should be able to synthesize proteins made sense to Schuman and others because
it was more economical and efficient. “It’s like the difference between
centralized and distributed freight shipping,” she said. “With central
shipping, you need a huge number of trucks that drive all over town, moving
freight from a central factory. But with distributed shipping, you have
multiple distribution centers that serve local populations, with far less
transport involved.”
Previous studies had
indicated that fragments of dendrites had the capacity to synthesize proteins.
Schuman and her colleagues believed that visualizing local protein synthesis in
living neurons would provide a more compelling picture than was currently
available.
The scientists began
their efforts to create a protein synthesis reporter by flanking the gene for a
green fluorescent protein with two segments of a gene for an enzyme called
calcium/calmodulin-dependent kinase II-α (CAMKII-α). By including CAMKII-α, the
researchers ensured that they could target their protein synthesis reporter to
dendrites.
In a series of
experiments, the scientists inserted the protein synthesis reporter into rat
neurons in culture, and then triggered protein synthesis using a growth factor
called BDNF. By imaging the neurons over time, they showed that the green
fluorescent protein was expressed in the dendrites following BDNF treatment —
unequivocal proof that protein synthesis was taking place. Going a step
further, the researchers showed that they could cause the fluorescence to
disappear by treating the neurons with a drug that blocked protein synthesis.
Schuman and her
colleagues also addressed whether proteins synthesized in the main cell body,
called the soma, could have diffused to the dendrites. “The soma is a
potentially greater source of proteins than the dendritic spines, so we had to
show convincingly that there was no way that the signal we were observing in
the dendrites was coming from the soma.” The researchers isolated the dendrites
by snipping them from the neurons. The isolated dendrites also exhibited
protein synthesis.
Schuman’s group then
created a version of the reporter that anchored the fluorescent protein to the
cell membrane. When they used laser light to “photobleach” the cell bodies to
eliminate any fluorescent protein, they still observed a prominent fluorescent
signal in the processes. This, too, indicated that protein synthesis was
occurring in the dendrites.
Intriguingly, said
Schuman, hot spots of protein synthesis were observed within the dendrites. By
tracking the location of the fluorescent signal over time, the researchers
could see that these hotspots waxed and waned consistently in the same place in
dendrites. “The main attraction of local protein synthesis is that it could
endow synapses with the capacity to make synapse-specific changes, which is a
key property of information-storing systems,” said Schuman. “The observation of
hot spots is consistent with this idea because it suggests that there are
localized spots of protein synthesis that are near synapses that may provide
new proteins to their associated synapses.”
Schuman and her
colleagues are now applying their protein synthesis reporter system to more
complex brain slices and whole mice. “In the whole animals, we’re exploring the
role of protein synthesis in information processing and animal behavior,” said
Schuman. “For example, in the hippocampus, the brain structure we study, most
neighboring neurons don’t seem to have functional similarities if you’re
recording signals from their cell bodies. Neighboring neurons appear to like
different kinds of stimuli. But using this protein synthesis reporter, we might
uncover similar functional domains in the dendrites of these different cells
that we wouldn’t see if we were just recording from their cell bodies,” she
said.
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