Older adults can still remember some of their childhood as though it happened yesterday. What keeps these memories around for a lifetime? This question has been shrouded in mystery, but scientists began to draw back the veil in 2006 by identifying a molecule in rats that maintains old memories. Now, researchers led by Yadin Dudai at the Weizmann Institute of Science in Rehovot, Israel, and Todd Sacktor at SUNY Downstate Medical Center, Brooklyn, New York, have taken this research a step further by showing that overexpression of the molecule, protein kinase Mζ (PKMζ), can strengthen memories long after they have formed. The research, reported in today’s Science, solidifies the case for the kinase having a unique role as a guardian of memory.
“This is an important paper,” said Oliver Hardt at McGill University, Montréal, Canada, who was not involved in the work. The data might inspire some scientists in the field to refocus their energy, Hardt suggested, shifting from work on memory formation and consolidation to a greater interest in long-term memory maintenance.
This week saw another advance in memory research as well, as a paper in today’s Cell describes an essential role for astrocytes in long-term memory formation. Researchers led by Cristina Alberini at Mount Sinai School of Medicine in New York City, and Pierre Magistretti at the University of Lausanne, Switzerland, found that lasting memories did not form unless astrocytes metabolized glycogen stores and released lactate, which was then taken up by neurons. The scientists showed that lactate was essential to stabilize long-term potentiation of synapses and trigger many of the downstream molecules that help build memories in neurons, although the mechanism remains mysterious.
Dudai and Sacktor’s work follows five years after the first discovery of PKMζ’s unexpected abilities. Sacktor and André Fenton at New York University showed that by administering an inhibitor of PKMζ in the insular cortex of rats, they could erase a conditioned taste aversion that had been learned three months earlier (see ARF related news story on Pastalkova et al., 2006). This was significant because PKMζ, an isoform of protein kinase C, is, to date, the only molecule that has been shown to sustain long-term, consolidated memories, scientists contacted for this story agreed. “Identification of PKMζ was a pretty big event,” Hardt said. “It was the molecule everyone had been looking for.” Science magazine hailed the finding as one of its 10 “Breakthroughs of the Year 2006.” Later work in the Sacktor lab showed that PKMζ not only acted in the insular cortex, but also in the hippocampus and neocortex (see Shema et al., 2007 and Shema et al., 2009), suggesting it is broadly active in the brain. One of the corollaries of the initial finding, Fenton said, was that “if PKMζ is really storing memories, then in principle, increasing PKMζ expression should improve memories.”
Dudai, Sacktor, and colleagues tested this hypothesis by designing lentiviral vectors to express either PKMζ or a dominant-negative version of the kinase. First author Reut Shema microinfused the viruses into the insular cortex of rats six days after he trained the animals to develop a dislike of a new taste by pairing it with lithium chloride, which makes rats sick. He chose this time period because by six days after training, memory is fully consolidated in rats (see Rosenblum et al., 1993). When the animals were tested seven days after viral infusion, the dominant-negative molecule had erased the learned aversion, just as the PKMζ inhibitor did in earlier experiments.
Taste aversion is normally a very strong memory, incapable of showing further strengthening, so for the trials of functioning PKMζ, Shema and colleagues produced a weaker version of the memory by adding much lower concentrations of lithium chloride to the new taste. The scientists found that administering PKMζ either a week before or a week after taste training significantly strengthened this weak memory. Not only that, but the more PKMζ that was expressed in the cortex, the stronger the memory became, showing a direct relationship between the kinase and memory intensity.
“This research adds, in a very significant and different way, to the evidence that PKMζ is a mechanism for storing memories,” Fenton concludes. He was not involved in the current paper. The burning question now is how the kinase produces these effects. Recent research has shown that PKMζ speeds up the transport of GluR2 subunits of AMPA receptors, Fenton said, probably by phosphorylating the trafficking machinery (see Migues et al., 2010). The upshot is that when PKMζ is present at a synapse, AMPA receptors are inserted at twice the normal rate, Fenton said. This results in twice as many AMPA receptors at the synapse, more uptake of glutamate, and stronger synaptic responses. When the kinase is inhibited, Fenton said, the AMPA insertion rate falls, and endocytosis catches up and trims the synapse strength back to baseline levels.
The model suggests that PKMζ should act only on synapses that are somehow “tagged” as part of a memory. Some research does show that the kinase accumulates preferentially in tagged synapses, where it maintains their long-term potentiation (see Sajikumar et al., 2005). In their overexpression experiments, Dudai and colleagues saw that only some dendritic spines contained PKMζ (see image below), lending further support to the idea that PKMζ is specifically sorted to tagged synapses. In ongoing work, Dudai said, he is seeking to test this hypothesis and discover how such a targeting mechanism might work. Dudai would also like to identify which subclasses of neurons are most affected by PKMζ overexpression, by using viruses that target gene expression into specific neuron types. Kurt Haas at the University of British Columbia in Vancouver notes that this model implies that PKMζ would have to remain in tagged synapses for the lifetime of a memory. “It is a fascinating shift in paradigms of thinking about how memories are stored,” he said.
Protein kinase Mζ (red) accumulates in some dendritic spines (yellow), but not in others (green). This supports the idea that PKMζ specifically targets synapses that have been marked for memory storage, although the hypothesis remains to be proven. Image credit: Weizmann Institute of Science, Rehovot, Israel
Dudai said that he has no current plans to test his PKMζ vectors in Alzheimer’s disease mouse models, because his immediate focus is to understand how the kinase works. Nonetheless, he believes that the protein might have an important role in cognitive disorders, and that this could be a fruitful line of research. Hardt suggests that the PKMζ findings might help shift scientists’ focus from treating the pathological symptoms of memory disorders to repairing or improving the machinery that maintains memory. Fenton notes that PKMζ and GluR2 subunits have been shown to build up in neurofibrillary tangles (see Crary et al., 2006)—an intriguing finding that implies PKMζ trafficking could be perturbed in AD and other dementias. The unknown mechanism that causes tangles to soak up the kinase might make a promising therapeutic target, Fenton suggested. Nonetheless, Fenton cautions it is too early to consider PKMζ overexpression itself as a treatment approach for cognitive disorders, because there is still so much basic research to be done before scientists understand what this molecule is doing.
One of the central problems for developing therapies, Fenton said, is that researchers still do not understand what memory is at a molecular level. Hardt takes it a step further, pointing out that not only do scientists not understand what memory is, but they know even less about forgetting. "What does a synapse look like before and after forgetting?" Hardt asked. Is a forgotten memory irretrievable? No one knows, but perhaps further study of PKMζ will shed light on this mystery.
In the Cell paper, Alberini, Magistretti, and colleagues took quite a different approach to the problem of memory, examining the role played by astrocytes. These glial cells, which outnumber neurons, were once considered mere second-string support players to the neuronal stars. In recent years, however, research has shown that astrocytes are much more essential to neurons than was thought, for example, having crucial roles in promoting synapse formation and elimination (see ARF related news story on Christopherson et al., 2005 and ARF related news story on Stevens et al., 2007).
Astrocytes are also metabolically coupled to neurons. These glia can break down glycogen energy stores, while neurons cannot. It is believed that neurons soak up the lactate released by astrocytes and use it as fuel. Neuronal activity stimulates astrocytes in turn, leading them to break down glucose (see Pellerin and Magistretti, 1994) and produce lactate (see Fray et al., 1996; Urrila et al., 2004).
To find out if this metabolic crosstalk was crucial to memory formation, first author Akinobu Suzuki began by confirming that lactate levels rose in the hippocampus of rats after they were trained to avoid a foot shock. When Suzuki and colleagues pharmacologically inhibited glycogen breakdown either immediately before or after training, lactate levels did not rise, as might be expected. This condition wiped out the rats’ long-term memory, although their short-term memory remained fine. The researchers rescued memory formation by injecting lactate into the hippocampus before training. The results suggested that lactate release is, in fact, essential for long-term memory formation.
At a mechanistic level, Suzuki and colleagues found that when glycogen breakdown was inhibited, long-term potentiation (LTP), a form of synaptic strengthening that is essential for learning, initially developed but did not last. Lactate injections rescued LTP. The authors also knocked down the levels of the lactate transporters MCT1, MCT2, and MCT4 using antisense oligonucleotides. For MCT1 and MCT4, reported to be enriched in astrocytes (see Pellerin et al., 2005), knockdown disrupted long-term memory, while lactate injection rescued it. Knockdown of MCT2, found in neurons, eliminated memory but lactate did not rescue it. The results fit with a model in which lactate must be transported out of astrocytes and taken up by neurons for learning to occur. Suzuki and colleagues also showed that MCT1 expression increases in response to learning; when they knocked down MCT1, long-term memories did not form. Finally, memory consolidation is known to require changes in numerous neuronal molecules, such as induction of activity-regulated cytoskeletal protein and phosphorylation of the transcription factor CREB and of cofilin, which is involved in remodeling the cytoskeleton. The authors showed that these changes were also blocked by glycogenolysis inhibition and rescued by lactate.
The million-dollar question now, Alberini said, is, What is lactate doing in neurons? It is possible that lactate is simply providing energy. However, the authors found that glucose injections only weakly and transiently rescued memory formation, suggesting that merely supplying an energy source is not enough to produce these effects. Alberini said the next step will be to determine how lactate is acting on neurons. She is also interested in examining animal models of cognitive impairment for defects in lactate production. Additionally, Alberini would like to look at brains from AD patients, measuring the expression of lactate transporters and astrocytic markers to see if this metabolism is disturbed.
Dmitri Rusakov at University College London, U.K., wrote to ARF that the identification of lactate’s role in memory formation is exciting. The findings also suggest that adding lactate could rescue memory impairment caused by faulty glycogenolysis, he wrote, but it remains to be seen how common this mechanism is in memory disorders (see full comment below).
Ben Barres at Stanford University in Palo Alto, California, noted in an e-mail that recent gene expression data from large array studies of purified cell populations have cast doubts on the older lactate transporter expression data (see Cahoy et al., 2008 ; Doyle et al., 2008; Dougherty et al., 2010). For example, the newer data indicate that MCT4 is not expressed in astrocytes, and MCT2 is not exclusive to neurons. Noting also that antisense knockdown does not eliminate expression, Barres suggested that the next step should be to use more specific molecular perturbations in defined cell populations to nail down the contributions of the different transporters.
For her part, Alberini said the most important thing is for researchers to enlarge their frame of thinking to include not just neurons but other cells as well. “Before, we were thinking about cognitive functions as a result of neuronal network mechanisms, but we now have to think about the crosstalk between astrocytes and neurons,” she said.—Madolyn Bowman Rogers
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