5 January 2013. Research has turned up many molecules that contribute to the formation of memories, but a single peptide, protein kinase M-ζ (PKM-ζ) has been hailed as the key player in maintaining them. Now, new research casts doubt on that starring role. In the January 2 Nature, back-to-back papers report that PKM-ζ knockout mice learn and remember just as wild-types do. They also have normal synaptic plasticity. The two groups, one led by Richard Huganir at Johns Hopkins University, Baltimore, Maryland, and the other by Robert Messing at the University of California, San Francisco, made different knockouts but obtained similar results. Researchers in the field agree that this means the kinase may not be essential for memory maintenance, but they differ on the exact interpretation. Several scientists Alzforum contacted believe that PKM-ζ may still be the main player in normal mice, but when the gene is deleted, cells recruit redundant pathways to compensate for its loss. Other researchers are more inclined to sideline the kinase, suggesting that alternate memory pathways may take center stage. This is an active area of research in many labs, and future studies will likely clarify the issue.
“The message of these papers is not that PKM-ζ is not important, but rather that it is one molecule in the complex system that plays a role in memory maintenance,” Paul Frankland at the Hospital for Sick Children, Toronto, Canada, told Alzforum. Together with colleague Sheena Josselyn, he wrote an accompanying Nature commentary on the findings.
The discovery in 2006 that an inhibitor of PKM-ζ could erase memories “turned the field on its head,” according to Kurt Haas at the University of British Columbia, Vancouver (see ARF related news story on Pastalkova et al., 2006). The constitutively active kinase was the first and still the only molecule shown to be crucial for sustaining long-term, consolidated memories. Science magazine named it one of its 10 “Breakthroughs of the Year 2006.” Later work by one of the authors, Todd Sacktor at SUNY Downstate Medical Center, Brooklyn, New York, demonstrated that overexpression of PKM-ζ can strengthen previously formed memories in rats (see ARF related news story).
Huganir and colleagues wanted to further investigate PKM-ζ’s effect on synapses. Co-first authors Lenora Volk and Julia Bachman knocked out the gene in mice. To their surprise, hippocampal slices from the animals exhibited normal long-term potentiation (LTP). The knockouts also performed normally in fear conditioning and spatial memory tasks. Also unexpectedly, the authors found that the PKM-ζ inhibitor (ζ inhibitory peptide, or ZIP) quenched LTP in hippocampal slices from PKM-ζ knockouts just as well as it did in wild-types, showing that ZIP can act through pathways other than PKM-ζ to disrupt synaptic plasticity. “This indicates that PKM-ζ is not the normal target for ZIP and is not mediating the effect on memory,” Huganir told Alzforum. “Now it’s up to the field to figure out what ZIP is doing.”
In the second paper, Messing and colleagues describe the generation of a different PKM-ζ knockout, which also behaved normally in fear conditioning, object recognition, motor learning, and drug reward memory tests. The only difference was that these knockouts appeared less anxious than wild-type mice. Similar to the Huganir mice, ZIP still affected these animals. First author Anna Lee injected the inhibitor into the nucleus accumbens of the knockouts and found that it erased cocaine reward memory just as well as in wild-types.
What do these findings mean for PKM-ζ? The data are confusing, Haas told Alzforum. Previous work from more than a dozen labs worldwide suggests that PKM-ζ can play an important role in memory maintenance. In addition to the overexpression experiments and inhibition by ZIP, blocking PKM-ζ function with a dominant negative molecule or using a different inhibitor also wipes out long-term memories. However, overexpression and dominant negative experiments can produce non-physiological results, Haas noted. “My opinion is that it’s possible that PKM-ζ still plays a role under normal conditions, but I think that has to be reevaluated in light of these studies,” he said.
André Fenton at New York University, who coauthored the original PKM-ζ paper, believes that the previous work and the new findings do not necessarily contradict each other. “The logical possibility is that when you get rid of something fundamental like PKM-ζ, it’s compensated for,” he said. Other researchers agree. “It would make sense that the cell would have redundant mechanisms to maintain memories,” Daniel Alkon and Thomas Nelson at West Virginia University, Morgantown, wrote to Alzforum. Future work should focus on finding out what these backup mechanisms are, Sacktor suggested, adding, “The key thing is we have three different inhibitors of PKM-ζ and they all do what nothing else has ever done, which is to erase memory. I think the evidence is pretty strong that for normal animals, PKM-ζ is the main molecule [that maintains memory].”
The fact that ZIP still erases memory in the PKM-ζ knockouts intrigues researchers. What might the inhibitor be acting on? PKM-ζ belongs to the protein kinase C (PKC) family, and has a closely related isozyme, PKC-ι/λ. Using in-vitro assays, Messing and colleagues found that ZIP quieted PKC-ι/λ just as effectively as PKM-ζ. They then looked to see if PKC-ι/λ levels went up in the knockout mice, but saw no evidence of this by Western blot. Huganir and colleagues also found no sign of changes in PKC-ι/λ in their knockout mice.
Despite these negative findings, many commentators suggested that PKC-ι/λ may be compensating for the loss of PKM-ζ in the knockouts. PKC-ι/λ differs from its cousin in that it contains a regulatory sequence, but this portion can be cleaved to form constitutively active PKM-ι/λ, which is almost identical to PKM-ζ. PKM-ι/λ functionally compensates for missing PKM-ζ in other scenarios (see Kishikawa et al., 2008; Rodriguez et al., 2009), and like PKM-ζ, it becomes activated during LTP (see Kelly et al., 2007), noted Oliver Hardt at McGill University, Montréal, Canada. Fenton pointed out that levels of PKM-ι/λ are very low in normal brain, and suggested that the methods in the two Nature papers may not have been sensitive enough to detect the kinase. He is currently looking for changes in PKM-ι/λ in the same knockout mouse used by Messing.
To test these ideas, Huganir has made a PKC-ι/λ knockout and a double knockout of the ι/λ and ζ isoforms, which he will examine for memory defects and sensitivity to ZIP. He also plans to perform a biochemical screen to find other molecules that can bind the inhibitor. The results of these and other ongoing studies should help pin down the pathways that sustain our long-term memories.––Madolyn Bowman Rogers.
Volk LJ, Bachman JL, Johnson R, Yu Y, Huganir RL. PKM-ζ is not required for hippocampal synaptic plasticity, learning and memory. Nature. 2013 Jan 2. Abstract
Lee AM, Kanter BR, Wang D, Lim JP, Zou ME, Qiu C, McMahon T, Dadgar J, Fischbach-Weiss SC, Messing RO. Prkcz null mice show normal learning and memory. Nature. 2013 Jan 2. Abstract
Frankland PW, Josselyn SA. Memory and the single molecule. Nature. 2013 Jan 2. Abstract