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-a (CAMKII-a). By including CAMKII-a, 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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