Saturday, March 22, 2014

Memory on the knife's edge

Amyloid-type proteins may be essential for long-term memory, even as they risk destroying it.

As we learn about the genetic aspects of many diseases, one is prompted to ask.. how could susceptibility to such a conditions ever have evolved? Susceptibility to Alzheimers and other degenerative dementias is a case in point. If they are caused by amyloid, or prion-like proteins that aggregate excessively, thereby destroying their cells and tissues, then the obvious question is: what do they normally do, and why hasn't this tendency towards aggregation been more stringently policed by evolution?

A recent paper on memory in flies (reviewed) offers the theory that in the case of memory, proteins that aggregate are central to the ability to form long-term memories. Aggregation is a feature, not a bug, and the only problem would then arise when this system works too well, or with abnormal partners.

Following the general trend in brain science, memory is being studied as, and found to be, a physical phenomenon. As far as we know, it consists of the persistence of physical and functioning synaptic connections among neurons that are re-inforced by use. The vast mesh of synapses connecting neurons in the brain form engrams that can be read back out upon partial stimulation of the encoded pattern. And memories are quite resistant to brain degradation- when symptoms of Alzheimer's appear, vast amounts of brain tissue are already gone. On the other hand, a specific injury to a key area can instantly destroy a lifetime of memory, the ability to form new ones, or other functions.

How do synapses work? Neurons have to grow if we are to learn, and they have to selectively connect with their downstream partners, the dendrites, cell bodies, and axons of other neurons, via synapses. All these are molecular processes, and one can imagine that if we are to have durable memories of anything, that molecular process has to have the contradictory features of durability as well as reversibility. We forget most of what we have ever experienced, and our sleep seems to be some kind of clearance and consolidation process, keeping some, and throwing out the rest. However that works, the synapse is well-known to be the focal point of memory, and protein synthesis has long been known to be required for new synapses to be made.

The fruit fly, despite its lowly intellect, has been an excellent organism to study neuronal activities, and even cognition and learning. In this case, prior work has identified one protein, CEPB, as one that is essential to memory durability. Without it, synapses still form, but then die off within a day of a training regimen, corresponding to the fly's inability to remember what it was trained for.
"Interestingly, ApCPEB [name of this gene/ protein in the sea snail] and Orb2 [the gene name in the fly] form self-sustaining amyloidogenic oligomers (prion-like) in response to the neurotransmitters serotonin in Aplysia and octopamine or tyramine in Drosophila. More importantly, the oligomeric CPEB is required for the persistence of synaptic facilitation in Aplysia and for the stabilization of memory in Drosophila. These observations led us to propose that the persistent form of memory recruits an amyloidogenic oligomeric form of neuronal CPEB to the activated synapse, which in turn maintains memory through the sustained, regulated synthesis of a specific set of synaptic proteins."
A second protein/gene was also found in this process, called Erb2. Mutations in either Orb2 or Erb2 do not affect initial learning, but destroy the fly's ability to remember a day afterwards what it would otherwise have remembered, as shown by unmutated control flies. The two proteins bind to each other, and Erb2 promotes the oligomerization of Orb2, which it to say, it helps it form long strings or globs, as one sees in magnified, extreme form in amyloid plaques, tau tangles, and the other hallmarks of the Alzheimers and related diseases.

The present researchers add a little to our knowledge by finding that Erb2 performs this feat of altering the behavior of Orb2 by getting a third protein, Lim kinase, to phosphorylate Orb2. Phosphorylation is a very common way to modify and regulate the activity of proteins, so this is not shocking. It is also quite reversible, in this case by yet another protein, protein phosphatase 2A. Erb2 apprears to be regulated itself by the state of the synapse, so it is a leading candidate to transmit the establishment / maintenance signals from neighboring cells or firing rates to the activities of Orb2.

But what is the point of this interaction, phosphorylation, and oligomerization? Orb2 interacts with a variety of other proteins active in protein translation, and seems to promote the various translation events needed for building a synapse. "The Cytoplasmic Polyadenylation Element Binding (CPEB) proteins are a family of RNA binding proteins that regulate the translation and subcellular distribution of a specific set of cellular mRNAs in various cell types including neurons." This is the hypothesized mechanism by which the signals transmitted by Erb2 to alter the behavior of Orb2 end up growing and stabilizing the synapse.

What is their data? First, they do a general assay for proteins that stick to Orb2. This nets over 50 proteins, many of which are involved in translation, like eIF4E, EIF3-S4, etc. Others are specific to the synaptic location, like Snap25 an Vap33. And one was Erb2, which they also saw stablized one form of Orb2, a tip-off to an important function. Then they show that Erb2 promotes oligomerization of Orb2 in vitro as well as promoting its collection in tiny physical globs in fly neurons.


This is typical data, and not very user-friendly. But it shows that Erb2 (here called Tob) and Orb2 interact, and that this interaction is promoted by neuronal activity, and that the resulting Orb2 protein complexes are amyloid-like. They used an antibody to Erb2 to collect anything sticking to it in a soup made from ground-up fly heads. They had used the chemical tyramine (top) to put the flies into neuronal overdrive before killing them. The matrix is full of controls: with tyramine, without, and with the anti-Erb2(Tob) antibody, or without (using "pre"-immune serum lacking the antibody). Then they ran all the proteins out on an electrophoretic gel that separated them by size (biggest at top), and stained the result with a separate antibody that binds to the Orb2 protein (in fact two of them: versions against only one of its forms (Orb2b) or against both (Orb2a, b)). This way, they can see only the Orb2 that was "brought down" by the Erb2 protein.

You can see that the Orb2 protein comes in two forms- a small monomer, and the larger dimer and higher oligomers, as indicated at the 107 kDa (aka molecular weight) and higher levels. Part B shows the whole experiment redone while treating the collected proteins with 2M urea, which is denaturant that blows up any conventional protein complexes. But it does not dissociate these Orb2 oligomers; evidence that they are very strongly associated as is typical for amyloid-type proteins.

Similar work explores the phosphorylation of Orb2 by Erb2, and its regulation by the protein phosphatase 2A and Lim kinase. This phosphorylation dramatically alters the lifespan of Orb2, from 1 hour to 24 hours or more, which is again a sign that its amyloid-like character can be specifically regulated.
"PP2A, an autocatalytic phosphatase, is known to act as a bidirectional switch in activity-dependent changes in synaptic activity. PP2A activity is down-regulated upon induction of long-term potentiation of hippocampal CA1 synapses (LTP) and up-regulated during long-term depression (LTD). Similarly, Lim Kinase, which is synthesized locally at the synapse in response to synaptic activation, is also critical for long-term changes in synaptic activity and synaptic growth."


Finally, they show data for the theory that Erb2 (Tob) is specifically needed in vivo to support long-term memory, not short-term memory. The test is a little sad. They place male flies with unreceptive females. Normal males get the hint in a hurry, and quit courtship attempts within 5 minutes. And normal males remember that female and her brushoff for several days, as shown in the controls in the two graphs at left. Red is the trained males, with suppressed (learned lack of) courtship. The "UAS-TobRNAi" and "201:Gal4" are partial versions of the transgenic construct, which is fully present in the far-right graph, and which expresses a sort of anti-Erb2, (by way of RNAi), when engineered into these flies. When the genetic alteration is fully present (right), the males have totally forgotten their training at 24 and 48 hours.

So, while the steps are small, these researchers have dug up some evidence for the idea that a central molecular component of memory in fly neurons, which has relatives in all animals including humans, has a regulated interaction with signalling molecules upstream that induce it to form aggregates that resemble those formed far more extravagantly by the culprits in degenerative brain diseases like Alzheimers. These aggregates have the virtue of longevity, though how they act on their downstream targets remains rather vague. Which may explain why those proteins susceptible to aggregation have been kept around in evolution, despite their dire risk when aggregation gets out of hand.

One might well ask a follow up question whether this Orb2 protein resembles in its sequence any of those known to go pathogenic in brain disease. I think the answer is no, but it is known to interact with an actual amyloid protein APLP1 in the mouse system, which provides a logical connection to a role in creating the kind of goop / plaque that can run amok in our heads. What those amyloid proteins are originally there for remains unsolved, but probably relates pretty closely to this synaptic establishment and maintenance system.

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