The cytoskeleton: A target for memory-enhancing drugs

What with the current debate about the use of "smart" drugs by academics, I thought it pertinent to republish this old piece from January of last year, about a bacterial toxin which has been shown to enhance fear conditioning and spatial memory in mice.

In the late nineteenth century, the great Spanish neuroanatomist Santiago Ramon y Cajal suggested that memories might be formed by the strengthening of connections between nerve cells:

Cerebral gymnastics are not capable of improving the organization of the brain by increasing the number of cells, because it is known that the nerve cells after the embryonic period have lost the property of proliferation; but it can be admitted as very probable that mental exercise leads to a greater development of the dendritic apparatus and of the system of axonal collaterals in the most utilized cerebral regions. In this way, associations already established among certain groups of cells would be notably reinforced by means of the multiplication of the small terminal branches of the dendritic appendages and axonal collaterals; but, in addition, completely new intercellular connections could be established thanks to the new formation of [axonal] collaterals and dendrites.

This phenomenon, known to modern neuroscientists as long-term potentiation (LTP), is the prevailing theory of the cellular basis of memory. LTP was first observed in the mid-1960s by Terje Lomo, a Norwegian neurophysiologist, while investigating the role of the hippocampus in short-term memory.

Experimenting on anaesthetized rabbits, Lomo isolated a simple neural circuit from the hippocampus, and observed that stimulation of a region at one end of the pathway resulted in changes in the electrical activity of the region at the other end. Unexpectedly, however, he also noticed that an enhanced response could be elicited in one region if a train of high frequency electrical stimuli was first applied to the other.

Lomo had discovered a mechanism whereby electrical activity increases the efficacy of intercellular signalling in the nervous system. Memories are believed to be formed by the strengthening and reorganization of synaptic connections. These changes ultimately lead to morphological changes in the nerve cells involved. Remembering - and forgetting -  require the formation or elimination of dendritic spines, the tiny finger-like protruberances at which neurons receive signals from other cells.

Underlying these morphological changes is the actin cytoskeleton, a cellular scaffold that has a wide variety of cell functions. It is along the cytoskeleton that chromosomes are segregated prior to cell division, and along which newly-formed synaptic vesicles, filled with neurotransmitter molecules, are transported to the nerve cell terminal.

The cytoskeleton is composed of microtubules (made of tubulin molecules), microfilaments (made of actin molecules) and intermediate filaments. It is a highly dynamic structure - the length of individual microtubules or microfilaments within the cytoskeleton changes constantly, by the addition or removal of its component molecules at one end or the other (polymerization and depolymerization, respectively).

Carla Fiorentini and her colleagues at the Istituto Superiore di Sanita in Rome have discovered that a bacterial toxin which induces polymerization of cytoskeletal molecules can enhance memory and learning in mice. The toxin, called cytotoxic necrotizing factor 1 (CNF1), is a protein synthesized by the bacterium Escherichia coli, which exerts its effects by activating a group of proteins called Rho GTPases, a family of related proteins that regulate the polymerization of the actin cytoskeleton.

Fiorentini's group injected CNF1 directly into the cerebral ventricles of mice, and then examined its effects on the animals in a series of behavioural tests. The animals were first trained to associate an electric shock with a specific conditioned stimulus. This fear conditioning results the formation of associative memories, in which a brain structure called the amygdala is known to be involved. By associating a particular context with an electric shock, the mice learn to avoid particular situations in which they can expect to receive a shock.

It was found that fear conditioning was improved in mice injected with CNF1, suggesting that the animals' associative learning had been enhanced. The improved ability of CNF1-treated mice to find a hidden platform in a water maze showed that the toxin was also effective in enhancing the animals' spatial memory. These effects were observed only in those animals treated with CNF1, and not in mice injected with either saline or a recombinant form of CNF1, in which an amino acid residue essential for its function had been modified. In the mice treated with CNF1, the improved learning was observed up to 28 days after conditioning had taken place.

These results led the researchers to investigate the effect of CNF1 in vitro. Slices of tissue from the neocortices of mice were cultured and treated with either CNF1, recombinant CNF1 or saline. Fluorescence microscope imaging revealed an enrichment of the cytoskeleton, and morphological changes in the dendritic trees, of CNF1-treated cells, while electrophysiological recordings showed that CNF1 enhanced neurotransmission in the hippocampus.

Thus, the toxin induced LTP in the hippocampal cells. By activating RhoA and Rac1, CNF1 was inducing the polymerization of actin molecules, thus driving the re-arrangement of cytoskeletal microfilaments and the synaptic modifications thought to be essential for memory formation.

As well as providing evidence for LTP, the findings made by the Italian researchers identify the cytoskeleton as a possible target for memory-enhancing drugs. In theory, such drugs could also be used to alleviate the cognitive impairments exhibited by patients with conditions such as Alzheimer's Disease.

Diana, G., et al. (2007). Enhancement of learning after activation of cerebral Rho GTPases. Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0610059104. [PDF]

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Fascinating! Was there any speculation on how Escherichia coli might benefit from producing CNF1?

@Jope: from a paper here:

"Bacteria express a wide range of virulence factors, including protein toxins that have evolved to interact with eukaryotic cellular machinery in a precise way."

Importantly, not all E. coli strains produce CNF1, and it's acquisition is an important marker of "bad" E. coli:

"Although belonging to the normal human intestinal flora, E. coli becomes highly pathogenic following the acquisition of genes coding for virulence factors, one of which being CNF1."

It's effect is of direct importance to a bacterium because it hijacks small G-proteins and forces them to switch on permanently. The effect of this is highly context specific, and in the context of an epithelium - such as the intestinal wall - they have a striking effect on cytoskeletal behaviour:

"The Rho GTPases are pivotal in controlling the actin cytoskeleton architecture... The prominent ruffling activity promoted by CNF1 permits epithelial cells to behave as phagocytes... [allowing] the capture and engulfment of different types of particles, including bacteria. This aspect is of particular relevance for the bacterial pathogenicity since the [phagocytosis] may possibly represent the route of entry of CNF1-producing E. coli."

I'm not a bacteriologist, so I can't go into detail on the advantages for a bacterium normally so happy to swim in the gut lumen to suddenly find itself inside a cell - possibly has something to do with having your nutrients provided for you rather than having to process them yourself - but they should probably be weighed up against the problems visited upon any parasite that kills it's host.

Effects on the brain are largely coincidental - for reasons of efficiency the same pathway (i.e. the same small g-proteins) happens to do something radically different in neurons than what it does in an epithelium. Of course, on a molecular level it's not doing anything radically different - it still binds and degrades GTP, and it still regulates the axin cytoskeleton - but that's a nice illustration of the beauty of context-dependent effects.