September 21, 2001
Researchers Find Enzyme Crucial to Preservation of Memories
Using a technique to eliminate the function of one enzyme in a
restricted memory-related region in the brains of mice, researchers
have shown that the enzyme is important in consolidating long-term
memories.
According to the researchers, their experiments — which showed that
defects in a key biochemical signaling pathway were responsible for the
animals’ inability to improve their long-term memory in a series
of maze tests — constitute a powerful approach to understanding
molecules involved in learning and memory.
“We believe that further studies using this technique will allow us to dissect in greater detail the differential roles and interactions of these signal transduction pathways, and how they contribute to this very complex mechanism of memory consolidation.”
Susumu Tonegawa
In an article published in the September 21, 2001, issue of
Cell, Howard Hughes Medical Institute investigator Susumu
Tonegawa and colleagues at the Massachusetts Institute of
Technology and the Vollum Institute reported that elimination of the
enzyme, calcium-calmodulin dependent kinase (CaMKIV), in the forebrains
of mice had profound effects on signaling pathways in the brain and
learning behavior.
The scientists began their studies to clarify the enzyme’s
role in late long-term potentiation (L-LTP), the process by which
enduring memories are established through a mechanism of activating
genes that trigger protein synthesis. This protein synthesis, in turn,
alters the synapses — connections between neurons — and
“etches” permanent memory pathways.
“CaMKIV had been implicated in long-term memory pathways in
the past, but previous studies had involved global knockout of the
enzyme in the entire animal,” said Tonegawa. “Such
knockouts gave inconsistent results because they affected the whole
brain throughout development. We decided to use a technique to inhibit
the protein only in the forebrain, which is more involved in higher
brain function.”
Tonegawa said that other research groups had attempted to knock out
a protein called CREB, which is involved in turning on gene
transcription in L-LTP, and which was believed to be activated by
CaMKIV. The results of these studies were inconclusive, Tonegawa said,
because there appeared to be multiple forms of CREB that could
compensate for any knockout.
Tonegawa and his colleagues used a genetic technique that allowed
them to replace the normal CaMKIV with a “dominant
negative” mutant enzyme that would be produced only in the
forebrains of the mice. Dominant negative enzymes have all of the
characteristics of the functioning enzyme — such as an ability to bind
normally to other molecules — but they lack the ability to carry out
an appropriate enzymatic reaction.
The scientists first studied the molecular details of the lack of
CaMKIV activity in brain slices from the transgenic mice. They
discovered that the base level of CaMKIV activity in the mouse brains
was normal, but when chemicals were added that mimicked the conditions
of neuronal activity, as in memory formation, the enzyme function was
significantly lower. The brain slice studies also revealed that CREB
activation by phosphorylation in the transgenic mice was suppressed,
strongly implicating a role for the CaMKIV in normal CREB activation as
a result of neuronal activity.
Experiments with brain slices also revealed that the transmission of
nerve impulses in the transgenic mice was normal, except under
conditions mimicking protein-synthesis-dependent L-LTP.
“These results pinpointed for us the role of CaMKIV in the
protein-synthesis-dependent type of LTP,” emphasized Tonegawa.
“This is very important, because in the past people have
published studies implicating another enzyme, called protein kinase A,
in LTP. However, that enzyme was not specific to the
protein-synthesis-dependent type of LTP.”
With clear physiological evidence that they had specifically
disrupted the CaMKIV pathway, the researchers next tested how well the
transgenic mice could consolidate memories of a water maze. The mice
were placed in a pool of water made opaque by floating beads, and
required to find a platform submerged just beneath the surface. The
transgenic mice initially learned the task as well as normal mice, but
as training continued, they became significantly less able to find the
platform.
“Thus, while these mice have a normal ability to acquire
memories, they have problems converting those memories into a long-term
form,” said Tonegawa. However, he noted, maze experiments still
present problems in interpretation. “This training takes place
over a two-week period, so the memory acquisition and consolidation
processes are superimposed,” said Tonegawa. “Thus, it is
difficult to know whether the deficit is primarily in the acquisition
phase or the consolidation phase.”
In an additional set of experiments, the scientists compared normal
and transgenic animals’ ability to acquire and consolidate the
memory that involves associating a mild shock to the footpads to the
specific context of the chamber in which the shock is administered. In
these experiments, memory acquisition could be more clearly separated
from memory consolidation, said Tonegawa. These experiments
demonstrated that the CaMKIV-deficient mice could learn to associate
the shocks to the chamber context, but they had difficulties in
converting such memories to long lasting memories, said Tonegawa.
“Our conclusion from these tests was that the CaMKIV pathway
was primarily involved in memory consolidation and retention,” he
said.
Tonegawa noted that memory consolidation in the transgenic animals
was not completely extinguished, suggesting that there may be parallel
signaling pathways involved in consolidation, or that there may have
been incomplete knockout of CaMKIV activity.
“However, we believe that further studies using this technique
will allow us to dissect in greater detail the differential roles and
interactions of these signal transduction pathways, and how they
contribute to this very complex mechanism of memory
consolidation,” he said. “Also, we want to know which genes
are activated in this process and how these gene products helps
establish these long-term changes in synaptic strengths.”
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