The current issue of The Economist contains a special feature about RNA. One of the articles summarizes the recent advances that have made molecular biologists realize the significance of RNA; the other discusses Craig Venter's recent application to patent an artificial life form.

In the editorial that accompanies the feature, an analogy is made between biology and physics. The biological sciences before the recent discoveries about RNA are likened to physics before the discovery of sub-atomic particles:

Because of the lifestyle they lead, sea sponges do not need, and therefore lack, nerve cells, muscle cells and internal organs of any kind. However, researchers from the University of California at Santa Barbara now find that one species of sea sponge, called Amphimedon queenslandica, synthesizes many of the proteins that are essential for the cell-to-cell communication that takes place within nervous systems. These surprising findings, which are published in the open access journal PLoS One, therefore provide clues about how the first neurons may have evolved in the most ancient of animals.

Neurons are specialized to communicate with one another. The signalling between one nerve cell and another takes place at a structure called the synapse, a miniscule gap of about 40 nanometres found at the junction between adjacent cells. The gap itself is no more than a space across which chemical signals (the neurotransmitters) diffuse. The active elements of the synapse are the two apposed cell membranes - the pre-synaptic membrane, from which the chemical signals are released, and the post-synaptic membrane, which detects the signals and responds to them in the appropriate way by communicating to the interior of the cell.

Each neuron has regions of its cell membrane specialized for sending the chemical signals, and others specialized for receiving them. The signalling process is known to involve many dozens of proteins that are either embedded within, or located just inside, the membrane. Each type of protein has a specific role in the signalling process. At the pre-synaptic membrane, for example, neurotransmitter molecules are packaged in synaptic vesicles, which remain "docked" at release sites along the inside of the membrane until needed. When a nervous impulse reaches the nerve terminal, it causes the vesicles to fuse with the membrane and to release their contents into the synapse. Once a vesicle is emptied of its cargo, it goes through an elaborate process of recycling. Docking of vesicles is mediated by one family of proteins; fusion of the vesicles with the membrane is mediated by another protein family, and a number of different families of protein are involved in the vesicle recycling process.

As the neurotransmitters diffuse across the synapse, they bind to receptors, Some receptors are voltage-gated ion channels; these are barrel-shaped proteins with a central pore that spans the membrane and opens in response to binding of a transmitter molecule. When the channel opens, it allows the passage of a specific type of ion (sodium, say, or potassium) into or out of the cell. This is a typical response of a neuron - the ion movements change the voltage across the membrane, altering the pattern of signals generated by that cell. Other receptors do not contain pores but instead are specialized to interact with various other proteins inside the cell, in response to a signal. These interactions initiate biochemical reactions within the cell, or communicate the signal to the cell membrane so that the activity of a specific gene or set of genes is altered.

For synaptic signalling to be effective, it is crucial that all the proteins involved are organized correctly. At both the pre- and post-synaptic membranes, this organization is achieved by a scaffold of proteins called the pre- and post-synaptic densities (left, click to enlarge). These structures are a specialization of the cytoskeleton found just beneath the pre- and post-synaptic nerve cell membranes. The density is a highly complex network - in humans it contains perhaps several hundred different types of protein - which organizes the molecular machinery needed for a neuron to detect and respond to the chemical signals sent to it by adjacent cells.

The density also regulates the movements of the machinery within the membrane and the area immediately inside it, and, so, is a highly dynamic structure. The dynamic nature of the density is believed to be crucial the strengthening of synapses that occurs during memory formation. Shuffling of proteins within the post-synaptic density after an initial stimulus may, for example, lead to an increase in the number of receptors at a certain region of the membrane, such that the subsequent response to the same stimulus becomes more efficient. Thus the density is essential in maintaining the integrity of the synapse, and is a major player in the neuronal plasticity that underlies learning and memory.

In the new study, Kenneth Kosic and his colleagues analyzed the Amphimedon genome, and found that it contains 36 families of genes known to encode proteins of the post-synaptic density. So, even though it has no neurons, this sea sponge synthesizes an almost complete set of post-synaptic density proteins. A comparison of the DNA sequences from the 36 sea sponge genes with the homologous sequences from humans, Drosophila melanogaster (fruit flies) and Nematostella vectensis (a cnidarian with a simple nervous system, consisting of a loose network of nerves) revealed striking similarities between the genes in all four species. One gene, called dlg, encodes a crucial component of the post-synaptic density scaffold. The protein product of that gene contains a number of regions that form the protein-protein bonds that hold the scaffold together. The segment of the dlg gene encoding these binding regions was found to be highly conserved - the DNA sequences in the sea sponge gene were identical to the human sequences. This suggests that in the sea sponge these proteins interact in exactly the same way as they do in the human post-synaptic density.

Amphimedon has nearly all the components required to make a post-synaptic density; only a few of the human postsynaptic density genes are missing from the sea sponge's genome - those encoding ion channel receptors for the neurotransmitter glutamate. (These genes are, however, present in the cnidarian, which expresses them in its simple nervous system.) In sponges, the genes are expressed predominantly in the flask cells of the free-swimming larvae, where they may be involved in sensing chemical cues found in the organism's environment. Flask cells with post-synaptic densities may predate the first neurons. If so, the first synapses may have evolved from post-synatic densities in a process called exaptation, whereby a pre-existing structure is modified slightly to perform a new function. It is, however, also possible that flask cells evolved from simple neurons that lost some of their synaptic components.

Reference:

Sakarya O., et al. (2007). A post-synaptic scaffold at the origin of the animal kingdom. PLoS ONE 2 (6): e506. doi:10.1371/journal.pone.0000506. [Full text]

Related:

• We may have inherited our brain from an ancient worm
• Robo-salamander provides clues about evolution of vertebrate locomotion

A new study into the biophysical properties of a highly reflective and self-organizing squid protein called reflectin will inform researchers about the process of "bottom-up" synthesis of nanoscale structures and could lead to the development of thin-film coatings for microstructured materials, bringing scientists one step closer to the development of an "invisibility cloak."

The reflectin protein comes from the Hawaiian Bobtail squid, Euprymna scolopes, which is native to the Central Pacific ocean. E. scolopes leads a nocturnal existence in the shallow waters off the coasts of Hawaii and Midway Island. Like other cephalopods, this species can manipulate the sunlight falling upon it to produce rapid changes in colouration and bioluminescence in order to camouflage from predators and prey and, as was recently discovered, to communicate with each other.

These changes are produced by a number of neurally-controlled photonic structures found throughout the squid's body. One of these, called the bilobed light organ, houses bioluminescent bacteria of the species Vibrio fischera. The squid and the bacteria have a symbiotic relationship - in return for generating light, the bacteria receive nutrients from the squid. The bacteria colonize the hatchling squid and secrete a toxin called tracheal cytotoxin. This toxin, which is a small fragment of a bacterial cell surface protein called peptidoglycan, causes whooping cough and gonorrhea in humans. But in E. scolopes, it serves a more useful function - it acts in synergy with various other substances to regulate the development of the light organ. The entire surface of the squid's body can also be considered as a light organ, as it contains reflective tissues in the mantle. Unlike the bilobed organ, whose reflectivity is static, the organs in the skin mantle have variable reflectivity, changes in which can quickly camouflage the squid and even make it invisibile.

The light organ and the reflective tissues in the skin mantle consist largely of proteins called reflectins. These are encoded by at least six genes which appear to be unique to squid. Nearly 44% of the reflectin primary sequence (the string of amino acids encoded in the reflectin gene) is made up of aromatic amino acid residues and amino acids containing sulphur. Reflectins are insoluble, and are deposited inside the light organs as flat, stacked structures called platelets. The reflectin molecules in the platelets are organized irregularly, and the layers formed by the stack of platelets alternate between areas of high and low refractive indices, so that the stacks act as multilayer reflectors (see the image below on the right). Because of this structure, incident light is reflected and scattered in all directions. The rapid changes in colouration that are characteristic of squid are the result of rearrangements in the organization of reflectin deposits within the reflective tissues, which may occur as a result of post-transcriptional modifications to the protein. Because of their chemical composition and the way in which they assemble themselves, the reflectins have the highest refractive index of any known proteins.

In the new study, which is published in Nature Materials, Rajesh Naik and his colleagues at the Air Force Research Laboratory in Dayton, Ohio inserted the gene encoding reflectin 1a into Escherichia coli bacteria. The recombinant protein synthesized by the E. coli cells was isolated and purified, and the self-assembly of the molecules was investigated under different conditions. The photonic properties of the structures were then investigated, and transmission electron microscopy was used to examine the structures formed in detail.

Naik's group found that the reflectin molecules formed a number of different types of structure, depending on the conditions in which they were assembled. In solutions of very low concentration, the reflectin molecules spontaneously precipitated to form nanospheres with diameters of 50-1,000 nanometres (nm, billionths of a metre), while with higher concentrations they formed . At low concentrations in non-reducing conditions (that is, in the absence of spare electrons), the precipitated nanospheres were optically clear - light could pass unhindered through them. But in reducing conditions (with spare electrons available) precipitation led to the formation of filamentous structures. (These fibres different from the fibres associated with aggregation of abnormally folded proteins in neurodegenerative disorders such as Alzheimer's Disease and the transmissible spongiform encephalopathies, which are formed by crystallization and not by precipitation.) And when left at 4°C for several weeks, these filaments formed a webbed structure that assembled itself into ribbons.

The researchers then sought to process the recombinant reflectin protein into films and fibres. They used a technique called flow-coating: small amounts of protein solution were added onto a silicon wafer substrate, and the edge of a blade was used to spread the solution across the surface of the wafer. This cast a thin film of the protein across the surface of the wafer. By altering the concentration of the solution used, films of different thickness were formed. The thickness of the film was found to determine the wavelength of light reflected by it. For example, exposure to water vapour dramatically increased film thickness from ~120 nm to ~207 nm. As a result, the wavelength of light reflected by the film changed from 760 nm (which corresponds to red light) to around 400 nm, which gave rise to a blue reflectance. When the water vapour was removed, the film became thinner and then began to reflect red light once again. In this way, the researchers formed films that reflected every colour in the visible light region of the electromagnetic spectrum; They also made gradient films whose thickness differed, so that a rainbow of colours was reflected along the length of the film.

The reflectin solution on the silicon wafer substrate was then dipped into an ionic solution (i.e. one containing positively or negatively charged atoms) called BMIM. This resulted in the formation of striped patterns of reflectin protein on the wafers. The patterns had highly regular spacing which extended unblemished for distances of up to several millimetres. The researchers found that the spacing between the stripes depended upon the velocity of dipping - the greater the velocity, the smaller the space between each stripe. These striped patterns are what materials scientists call diffraction gratings - reflective or transparent elements that split incident light into its constituent wavelengths and are used in a variety of optical devices. Light reflection by the reflectin stripes was observed even while the silicon wafers were still submerged in the solvent, and the wavelength of light scattered could be changed by increasing or decreasing spacing between the stripes.

The researchers had provoked nature into initiating some of her own mechanisms of nanofabrication. The reflectin molecules assembled themselves into nanometre-sized spheres and striped microstructures with photonic properties that could be manipulated. Knowledge of how the self-assembly of reflectins can be manipulated will inform researchers who are trying to synthesize supramolecular nanostructures from the "bottom up". But a better understanding of these mechanisms and of the properties of reflectin has another potential application: the synthesis of small-scale materials for use in the development of invisibility cloaks.

To this aim, various research groups are using so-called metamaterials, whose nanoscale properties alter the way in which their surfaces reflect visible light. Objects are visible because light bounces off them; in theory, a material which could cause incident light to pass round it could be used in a cloak that covers objects and renders them invisible. The military is, of course, very interested in developing such a material. As the current research was partly funded by DARPA (the research and development arm of the Pentagon), the development of an invisibility cloak could well be the ultimate aim of the project.

References:

Kramer, R. M., et al. (2007). The self-organizing properties of squid reflectin protein. Nature Mater. doi: 10.1038/nmat1930. [Abstract]

Crookes, W. J., et al. (2004). Reflectins: The unusual proteins of squid reflective proteins. Science 303: 235-238. [Full text]

Now, Itay Baruchi and Eshel Ben-Jacob of Tel Aviv University show that networks of cultured neurons can also store information. The image on the left shows their experimental set up. Nerve cells were isolated and cultured on a specialized dish in which microelectrodes are embedded so that the electrical activity of the cells can be recorded. A micropipette was then used to apply picrotoxin to small groups of cells in specific locations on the culture dish. Picrotoxin is a GABA receptor antagonist; its addition to the culture dish therefore suppressed the activity of inhibitory interneurons in the cell culture. As a result, synchronized bursting events (SBEs) - waves of electrical activity with specific patterns in both space and time - were observed in the nerve cell culture.

The cultured neurons "stored" information about the patterns of electrical activity evoked in the network by application of picrotoxin. Several different types of SBEs were evoked in the network, each of which starts at a specific location in the culture dish and is propagated along a specific trajectory. This activity continued as long as picrotoxin was applied; when the drug was removed, the cultured cells returned to their basal activity. But the SBEs could be precisely reproduced later on. If the initial application of picrotoxin to a specified location on the culture dish generated a specific type of SBE, exactly the same pattern of activity could be elicited up to 40 hours later by applying picrotoxin to the same location. Thus, the collective activity of the cultured cells had somehow been "imprinted" within the network.

Reference:

Baruchi, I. & Ben-Jacob, E. (2007). Towards neuro-memory-chip: Imprinting multiple memories in cultured neural networks. Phys. Rev. E. doi: 10.1103/PhysRevE.75.050901. [Full text]

"Schematic representation of mammalian retina structure. Artistic grouping of cells and direction of current flow." A, layer of rods and cones; B, visual cell body layer; C, outer plexiform layer; D, bipolar cell layer; E, inner plexiform layer; F, layer of ganglion cells; G, optic nerve fibre layer; L, central fossa. Modified from a photograph taken from the original (22x33 cm). Drawn on sheet/ paper.
P. Y. 1901. Cajal Institute - CSIS - Madrid, Spain.

This diagram by Santiago Ramón y Cajal shows the neural circuitry of the vertebrate retina. The retina's inverted structure seems ill-suited to its function: the rods and cones (labelled A, a and b in the diagram) are the photosensitive cells that transduce light energy into electrical impulses; they point away from incoming light, and are located at the back of the retina, so that light entering the eye has to pass through several layers of randomly oriented and irregularly organized cells before it reaches them. The retina also contains nerve fibres that are positioned perpendicular to the path of light entering the eye, and many of the structures in the upper layers have a diameter similar to that of the wavelength of visible light. Because of this inversion, one would think that incident light entering the eye should be subjected to a significant amount of reflection and scattering. Yet, nature somehow contrived to overcome this awkward architecture, and the retina performs its function perfectly.

As well as the various types of neurons, the retina contains specialized glial cells called Müller cells, which are arranged in parallel to each other and are oriented in the direction along which light travels through the eye. Müller cells are about 150 µm (micrometres, thousandths of a millimetre) in length, and span the entire thickness of the retina, projecting from the vitreous humour (the viscous fluid in the back of the eye) to the back of the retina where light enters the rods and cones. Müller cells have, like other glial cells, been largely ignored until recently: they were thought to do little more than support and nourish retinal neurons. (Notice that Cajal's diagram does not show Müller cells.) But in recent years it has been determined that glial cells perform other important functions, and now, new research, published online in the Proceedings of the National Academy of Sciences, shows that glial cells may also be nature's solution to the inverted retina problem. The new study, led by Jochen Guck of Cambridge University and Andreas Reichenbach of the Paul-Flechsig Institute of Brain Research at the Universität Leipzig in Germany, provides evidence that Müller cells function as optical fibres that transmit light through the retina.

Guck and his colleagues dissected guinea pig retinae, placed them under a confocal scanning microscope, and used a light source to mimic the natural illumination to which the tissues would be exposed. They then scanned the back of the tissues to determine the patterns produced there by light entering the tissue. To their surprise, the images they obtained contained a regular pattern of bright spots which alternated with darker areas on which there was less light. Retinae from rabbits and humans showed the same reflection pattern. Serial sections were then cut from the back of the retina. The dark spots they had observed were found to be 2-3 µm in diameter and spaced 5-6 µm apart. When the sections were reconstructed, it was found that the dark spots formed tubular structures that ran the entire thickness of the retina, and that they had funnel-shaped structures with a diameter of approximately 15 µm at one end. The extent of back-scattering (that is, the scattering of light in the direction from which it came) in the different regions of the back of the retina was then examined. This showed that there was significant back-scattering in cell layers near the photoreceptors, but very little in the dark spots, indicating that the tube-like structures within the dark spots transmit far greater amounts of light than the surrounding tissues. The arrangement of tube-like structures was also found to correspond well to the size and spacing of Müller cells.

Fluorescence microscopic image of the retina, showing Müller cells stained red (left, kindly provided by Professor Andreas Reichenbach) and individual Müller cells in the dual beam laser trap (right).

The researchers then investigated the optical properties of the Müller cells. Guinea pig retinae were treated with enzymes that cause the cells in the tissue to dissociate from one another. A dual beam laser trap was then used to investigate the ability of Müller cells to propagate light. (Like optical tweezers, laser trapping is a technique by which beams of light are used to keep particles or cells in a fixed position.) Individual cells floating in a suspension were aligned between the ends of two optic fibres. One of the fibres could be used to shine light onto the cells; the other was connected to a power meter. By bringing the fibres into contact with a free-floating cell, and passing light from one fibre through the cell to the other fibre, the amount of light passing through the cell could be measured. It was found that the amount of power entering the output fibre was greatest when a Müller cell was aligned in the same orientation as the fibres; but when the cell was rotated or removed from the trap altogether, the power output decreased dramatically. A dye called MitoTracker Orange, which fluoresces when struck by light, was then injected into the Müller cells, enabling the researchers to visualize the path of light. This confirmed that light was indeed passing straight through the cells.

This elegant set of experiments shows that the Müller cells function as conduits which guide the passage of light through the tissue of the retina. The funnel-like structures observed are the endfeet of the Müller cells, which are densely packed and form a cobblestone pattern on the membrane closest to the vitreous humour of the eye. During the laser trap experiments, one of the fibres was misaligned so that, in the absence of a Müller cell, the light did not strike the second fibre; when a Müller cell was then placed between the two fibres, it captured the light from the first fibre and guided it towards the second. Thus, the endfeet seem to be crucial in capturing divergent rays of light and guiding them towards the photoreceptors at the back of the retina. They also have a lower refractive index than other parts of the Müller cell and other cells in the retina, and serve to minimize reflection of incident light as it passes from the vitreous humour into the uppermost layers of the retina.

The way in which Müller cells transport light is similar to the mechanism by which the optical fibres in fibre optic plates carry light. Fibre optic plates consist of optic fibres bundled together, and are used instead of lenses to transfer images between distant locations; this occurs without distortion of the image or loss of image detail. Müller cells may perform the same function in the retina; each one is coupled to one cone photoreceptor and (in guinea pigs and humans) ten rods; the Müller cell arrays could therefore faithfully transmit the pattern of light falling on the front of the retina to the photoreceptors at the back of the retina, thus minimizing distortion of the image. And, because the Müller cells are funnel-shaped and narrow, they take up only 20% of the space in the retina; this leaves plenty of room within the tissue for the neural circuitry.

Reference:

Franze, K., et al. (2007). Müller cells are living optic fibers in the vertebrate retina. PNAS doi: 10.1073/pnas.0611180104. [Abstract]

Imagine the mRNA to be like a long piece of magnetic recording tape, and the ribosome to be like a tape recorder. As the tape passes through the playing head of the recorder, it is "read" and converted into music, or other sounds...When a "tape" of mRNA passes through the "playing head" of a ribosome, the "notes" produced are amino acids and the pieces of music they make up are proteins.

Music is not a mere linear sequence of notes. Our minds perceive pieces of music on a level far higher than that. We chunk notes into phrases, phrases into melodies, melodies into movements, and movements into full pieces. similarly proteins only make sense when they act as chunked units. Although a primary structure carries all the information for the tertiary structure to be created, it still "feels" like less, for its potential is only realized when the tertiary structure is actually physically created.

Now, using a program called Gene2Music, you can transcribe any DNA sequence into music. The program, which was developed by molecular geneticists Rie Takahashi and Jeffrey Miller of the University of California, Los Angeles, uses an algorithm that converts each codon in the DNA sequence into a musical chord. Codons for hydrophilic amino acids (which are attracted to water) have a high key, codons for hydrophobic amino acids (which are repelled by water) have a lower key, and the duration of each chord is determined by the frequency of its corresponding codon within the transcribed DNA sequence.

Using Gene2Music, Takahashi and Miller have so far generated more than a dozen pieces of music, including transcripts of the huntingtin and cytochrome c genes. The aim of the project is to make the visualization of proteins easier for scientists, and to make molecular biology more comprehensible to non-scientists. Takahashi says it was inspired by a blind meteorology student and Cornell University, who devised a method by which the different colours on a weather map could be converted into musical tones.

This isn't the first time DNA sequences have been translated into music. In 1995, for example, the British band The Shamen collaborated with Ross King to produce a track called S2 Translation, which is based on the coding sequence for the 5HT-S2 receptor. The track was generated in a way that sounds similar to Takahashi's and Miller's method:

The number and nature of bass notes per codon/bar were determined by the hydrophobicity/hydrophilicity, ionic charge (positive or negative) and size of each amino acid residue (Proline, for example,which has no characteristics other than its small size, can be identified easily as the bars where the bass line 'drops out'). The musical output resulting from these rules was further processed by mapping the notes onto different tonalities, both to make the piece more interesting, and to suggest the organisation of the protein molecule into regions of different secondary structure (although since S2 is a membrane protein and thus impossible to crystallise outside the lipid bilayer, this was definitely creative licence).

Previous pieces of DNA music have tended to sound unmelodic, because they often contain jump distances of up to two octaves (16 notes) from one tone to another. Takahashi and Miller overcame this by assigning three notes to each codon. With a triad chord for each codon, the differences between successive chords in the music are reduced.

Professor Li-Huei Tsai and her colleagues developed a transgenic mouse model of Alzheimer's Disease, in which expression of a gene called p25 gene, which has been implicated in various neurodegenerative diseases, induces extensive neuronal cell death in spatially restricted regions of the forebrain. p25 expression can be induced at any point during the life of the transgenic animals by supplementing their diet with a chemical called doxycyclin.

Mice expressing the p25 transgene were trained to perform two memory tasks. One task tested the animals' associative memory, the other tested their spatial memory. For the associative memory task, the mice were classically conditioned to fear a compartment in the enclosure in which they were kept. After repeatedly receiving mild electric shocks when placed in the compartment, the mice learnt to associate the two; afterwards, when returned to the compartment, they would freeze instead of exploring their environment, indicating that they were fearful of receiving a shock. The mice were also trained to perform a spatial memory task in which they find a platform submerged in murky water. When the animals reached 11 months of age, expression of the p25 gene was induced for a period of up to 6 weeks; as a result, the animals' performance on both memory tasks was severely impaired.

The mice were then returned to their cages. The researchers added toys, running wheels and various other stimuli to the cages of one group of mice. The toys were changed on a daily basis for four weeks. Nothing was added to the cages in which the control mice were kept (the "home" cages). The performance of both groups of mice in the two memory tasks were then re-tested. It was found that the mice exposed to the enriched environment performed significantly better on both memory tasks than the control animals. When placed in the compartment that they had previously associated with an electric shock, the mice that had been kept in an enriched environment quickly froze, indicating that they once again remembered the association, and, when tested on the spatial memory task, they successfully located the submerged platform. In contrast, the control animals did not freeze when placed in the compartment in which they had received the electric shocks, and could not locate the submerged platform.

The researchers then examined the brains of the mice. First, the brains of both groups of animals were weighed, and it was found that there was no significant difference between the weight of the brains of both groups. Thus, the observed memory recovery occurred despite extensive neuronal death and loss of synapses. This suggests that rather than being "lost" altogether, the memories persist, so that it is retrieval, and not the encoded memories themselves, that are impaired by neurodegeneration. Antibody staining was used to compare the distribution of a number of synaptic in the two groups of animals. This showed that, in the brains of mice kept in an enriched environment, levels of the proteins MAP-2 and synaptophysin (which are both involved in synapse formation) were markedly increased in the hippocampus, which is known to be involved in the encoding of memories, and anterior cingulate cortex, which has been implicated in the consolidation of long-term memories. The data suggest that the enriched environment led to memory recovery by re-establishing the synaptic networks, and not by inducing growth of new nerve cells or nerve cell processes. (Comparison of MAP-2 expression in the CA1 region of the hippocampus in experimental and control animals is shown in the image at the top, from the Supplemental information accompanying the paper.)

Proteins called histones were then compared in the brains of the two groups of mice. Histones are closely associated with DNA - they act as spools around which DNA is tighly wrapped. A chromosome consists in equal parts of a single DNA molecule and histone proteins, which together are termed chromatin. Histones play a vital role in regulating gene expression; chemical modification of histones - by, for example, the addition or removal of acetyl (-COCH₃) or methyl (-CH₃) functional groups - causes the chromatin structure to open or close, so that the information contained within the DNA is made more or less accessible to the enzymes involved in protein synthesis. When Tsai's group used antibody staining to examine the chromatin in their animals, they found that there was increased histone acetylation (addition of acetyl groups) in the hippocampi and cortices of mice that had been exposed to the enriched environment compared to the control animals. This was observed as little as 3 hours after exposure to the enriched environment. The enriched environment appeared to restore memory in the animals by remodelling the chromatin structure, thus enabling expression of the genes involved in the synaptic plasticity underlying memory.

Having established that the enriched environment leads to opening of the chromatin structure by acetylation, the researchers then sought to determine whether or not a drug that prevents removal of acetyl groups from histones could also restore memory. Another group of groups of animals was trained to perform the memory tasks before p25-mediated neurodegeneration was induced. One group of animals was then injected with sodium butyrate, a histone deacetylase inhibitor. The compound led to a significant improvement in the performance of the mice on the memory tasks, as evidenced by reduced freezing behaviour in the associative memory task and successful location of the submerged platform in the spatial memory task. Examination of the animals' brains showed that the compound had exactly the same effect as exposure to an enriched environment - increased levels of the synaptic proteins MAP-25 and synaptophysin in the hippocampus and anterior cingulate cortex.

It has long been known that gene transcription is necessary for memory formation, but it is only very recently that epigenetics - changes in gene expression not linked to changes in the DNA sequence itself - has been implicated in memory. The findings of Tsai's group therefore confirm the role of epigenetics in memory formation. They also implicate histone deacetylase enzymes as a potential new target for drugs to treat Alzheimer's Disease and other neurodegenerative diseases in which memory is impaired; there is, however, no indication as yet that histone deacetylase inhibitors would have a beneficial effect in humans. And, while most researchers trying to develop treatments for such conditions have focused on the early stages of the disease process, with the aim of slowing or halting all together disease progression, these findings suggest that mental stimulation could slow, or perhaps reverse, the memory impairment that occurs at later stages.

Reference:

Fischer, A., et al. (2007). Recovery of learning and memory is associated with chromatin remodelling. Nature doi: 10.1038/nature05772. [Abstract]

Related:

• Eternal erasing of the scary memory
• A possible target for memory-enhancing drugs
  

Reference:

Watson, J. D. & Crick, F. H. C. (1953). A Structure for Deoxyribose Nucleic Acid. Nature 171: 737-738. [Full text]

For geneticists, who are concerned with the gene as a unit of inheritance, that definition is fine. But for molecular biologists, who study the physical processes by which information in the DNA is used to synthesize proteins, it is inadequate, because in recent years, they have discovered, among other things, that the coding sequences of some genes overlap with those of others, and that parts of the coding sequences for some proteins are separated by large distances or are actually found on different chromosomes. Nevertheless, lets rub those chromosomes, and see what genes the genie has for us in this edition.

First of all, Hsein-Hsein Lei provides details about a DNA test for Type 2 diabetes at Genetics & Health. Type 2 (or non-insulin dependent) diabetes, is a metabolic disorder that affects up to 100 million people worldwide. It occurs later in life, when the body becomes resistant to insulin, the hormone that normally mediates the uptake by cells of glucose from the bloodstream, leading to high blood sugar levels (hyperglycaemia). The gene being tested for illustrates nicely the inadequacy of the conventional definition of the gene - its coding sequence lies entirely within that of a gene encoding the transcription factor 7-like 2 protein. By the way, people with type 1 diabetes need to inject themselves with insulin regularly to maintain their blood sugar levels. The insulin gene was one of the very first genes to be cloned. Researchers from Genentech, the world's first biotechnology company, generated a recombinant insulin gene that they expressed in bacteria. This eventually enabled the synthesis of insulin on an industrial scale, for the benefit of those suffering from type 1 diabetes.

Another condition for which genetic testing is available is Huntington's Disease (HD), an inherited, progressive neurodegenerative disorder characterised primarily by involuntary movements. These symptoms are caused by the death of neurons in the basal ganglia, a set of subcortical brain structures involved in the control of movement. One of the genes involved, huntingtin, is found on the short arm of chromosome 4, and the condition is inherited in an autosomal dominant manner. HD is a trinucleotide repeat disease; at one end of the gene, a sequence of 3 DNA bases are repeated multiple times, and mutations in the huntingtin gene cause an increase in the number of repeats. If the number of repeats reaches 40 or above, a mutated form of the Huntingtin protein is synthesized; this mutant protein is somehow involved in the neuronal cell death. Their is a direct correlation between the number of trinucleotide repeats and age of onset of HD - the greater the number of repeats, the earlier the age of onset. Leslie, who writes a blog called Paternal Age and De Novo Single Gene Disorders etc., has submitted the abstract for a 1993 paper about the association between Huntington's disease mutations and advanced paternal age.

Larry Moran, author of the Sandwalk blog, gives us two posts about blood clotting, a very elegant process that occurs as a result of an intricate biochemical cascade involving more than a dozen proteins. At the heart of the clotting process are two proteins, called fibrinogen and thrombin. Fibrinogen contains a number of adhesive regions which are usually covered up. Thrombin is a protease - an enzyme that snips other proteins. Following an injury, thrombin acts on fibrinogen molecules to expose their adhesive regions, causing them to form an insoluble clump that stems blood flow at the site of injury. In organisms with a simple circulatory system containing relatively low volumes of blood flowing at a low temperature, these two proteins are all that is needed for the clotting reaction. But in the vertebrate lineage, blood flows through its vessels under high pressure and, during the course of evolution, a series of gene duplications resulted in an increased number of clotting factors; as a result, there was a massive amplification of the clotting process, such that a more rapid and efficient response can be generated after the initial stimulus. Larry's first post is about a number of human anticoagulant genes implicated in various cardiovascular conditions, while the second is about anticoagulants, the chemicals which prevent blood clotting. Larry also submitted a third post, about human genes for the pyruvate hydrogenase complex. The products of these genes form a large enzyme complex that catalyzes a very important reaction - the conversion of pyruvate to acetyl-Coenzyme A, a molecule involved in many biochemical reactions that are crucial to the cell.

We'll finish with general genetics posts and posts about genes in organisms other than humans. Over at ScienceRoll, Bertalan has two posts; one about genetics in Second Life, the three-dimensional virtual world recently built by internet users, and another containing a slideshow of a recent presentation he gave on genetics and Web 2.0. Grrl Scientist, who blogs at Living the Scientific Life, has a post about a mouse model of bipolar disorder (also known as manic depression). Individuals who suffer from this condition experience dramatic mood swings, in which they cycle between episodes of euphoria and hopelessness. It's not surprising, then, that a number of the genes implicated in bipolar disorder are expressed in the suprachiasmatic nucleus, where they regulate the circadian rhythm. At Ouroboros, Chris has a post about Sirtuin-2, one of a family of proteins that have been implicated in the extension of life span associated with calory restriction. This phenomenon has most extensively studied in yeast cells and nematode worms, but there are also mammalian versions of the sirtuin genes.

At Sciencesque, Tim discusses the sequencing of the Tyrannosaurus rex collagen protein. This story was big news recently; the surprising similarity between the T. rex protein sequence and that of chickens led most commentators to state that barbequed T. rex would have tasted like chicken! Finally, my contribution is about the evolution of dorso-ventral (D-V) patterning in the developing nervous system. This process is regulated by a highly evolutionarily conserved family of proteins called the BMPs (bone morphogenetic proteins). Research published this week shows that the mechanisms of D-V patterning in the marine ragworm, a "living fossil" thought to most closely resemble the common ancestor of vertebrates, worms and insects, occurs in a very similar way to, and involves exactly the same molecules as, D-V patterning in the zebrafish, which is a vertebrate. The implication is that vertebrates (including humans) may have inherited the organization of their nervous systems from an ancient worm.

That's it for this edition of Gene Genie. The next one will be on 5th May at ScienceRoll. You can submit your genes here.

The method uses a protein called halorhodopsin, which was recently isolated from Natronomas pharaonis, an extremophile archaebacterium that thrives in highly salty conditions. Halorhodopsin is a light-activated chloride channel, which opens in response to green-yellow light, allowing an influx of chloride ions. Boyden and his colleagues fused the halorhodopsin gene to the gene encoding green fluorescent protein (GFP). The construct was under the control of the CAMKII promotor, a regulatory DNA element which drives gene expression in neurons of the mammalian forebrain. Using a lentivirus vector, Boyden's team shuttled the construct into cultured rat hippocampal neurons; cells expressing the construct could be easily visualized because they fluoresced green.

Microelectrodes were used to inject ionic solutions into the cells; the electrical currents evoked trains of up to 20 action potentials at a frequency of 5 per second. At the same time, pulses of light were delivered to the cells, at specific phases in the trains of action potentials, to inhibit the activity of the cells. The light activates the halorhodopsin molecules, which then pump negatively charged chloride ions into the cell. This hyperpolarizes the membrane of the nerve cell, i.e. increases the difference in voltage between the inside and outside of the membrane, so that the cell is less likely to generate a nervous impulse. The induced hyperpolarizations occurred within 15 milliseconds of the light pulse, and were reversible - the currents were deactivated within 15 milliseconds of cessation of the light pulse. This temporal resolution is so high that the light pulses could abolish single spikes in the train; a pulse timed to coincide with the 17th action potential in the train eliminated that spike from the train, but left spikes 16 and 18 unaffected.

Boyden's team assayed the effects of the construct on cultured cells. It was found that, in the absence of light, the electrical properties and basal activity of the transfected cells appeared to be no different from wild-type (normal) cells. Expression of the halorhodopsin-GFP construct remained stable for up to one week and did not lead to cell death.

In previous work by the same group, a similar technique was used to activate neurons. In this case, the protein used was channelrhodopsin-2 (ChR2), a light-gated ion channel from the green alga Chlamydomonas rheinhardtii. In transfected rat hippocampal cells, ChR2 is also expressed robustly, and is observed to be localized at the cell membrane for weeks after transfection. Activation of ChR2 by pulses of blue light generates inward currents of protons (hydrogen ions). These currents are induced even more rapidly than those mediated by halorhodopsin - within 1 millisecond of onset of the light pulses. The currents depolarize the cells, i. e. they bring the membrane voltage nearer to zero, inducing action potentials; rapid light pulses delivered in quick succession evoke realistic trains of action potentials.

In the PLoS One paper, Boyden's group describe combining the two systems by transfecting cells with both constructs, so that they simultaneously expressed halorhodopsion and ChR2. In these cells, alternate pulses of yellow and blue light induce hyperpolarizations and depolarizations of targeted neurons, respectively, providing a method for ultrafast, rapidly reversible and extremely precise control of neuronal activity. The high spatiotemporal resolution of the technique provides the fastest control yet of neuronal membrane voltage, enabling the electrical activity of the neurons to be controlled without disturbing the normal firing patterns of the cells.

Optical inhibition and/ or activation of neurons will prove to be a powerful tool for researchers. It will, for example, enable them to introduce temporary functional lesions in specific cells or groups of cells. By silencing specific subsets of cells for periods of less than a second during the performance of a task, the role of the timing of action potentials in neural computation can be investigated. Advances in in vivo imaging techniques, such as two-photon fluorescence microscopy, should enable researchers to photoactivate cells within intact tissues. Eventually, the technique could also lead to the development of optical neural prostheses for controlling the aberrant electrical activity associated with conditions such as Parkinson's Disease and epilepsy. Boyden and his colleagues plan to start experimenting with such devices in mice later this year.

References:

Han, X. & Boyden, E. S. (2007). Multiple-color activation, silencing and desynchronization of neural activity, with single-spike resolution. PLoS One 2: doi: 10.1371/journal.pone.0000299. [Full text]

Boyden, E. S., et al. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8: 1263–1268. [Full text]

Edward Callaway and his colleagues at the Salk Institute's Systems Neurobiology Laboratory, together with collaborators from the Max von Pettenkofer Institute and Gene Center at Ludwig Maximilians University in Munich, Germany, have developed a transsynaptic tracer which enables them to examine neuronal connectivity far more accurately than existing methods. The tracer, which is based on the rabies virus, can be targeted to individual neurons, and spreads only to those cells which form direct synaptic connections with it. The work is reported in the current issue of Neuron.

Rabies was chosen because, like herpes viruses, it infects neurons in the peripheral nervous system and is then transported along the nerve fibres to enter the central nervous system. It is less damaging to neurons, but infects them far more efficiently, than herpes viruses, which also target peripheral neurons and which are commonly used for tracing neuronal connections. The intact rabies virus is non-specific in its infection of peripheral neurons, and expresses a surface glycoprotein that is required for it to spread from one cell to another.

In order to use it to infect specified neurons, Gallaway's group constructed a modified version of the rabies virus. The construct contained a deletion mutation - the surface glycoprotein was removed, and replaced with the gene encoding green fluorescent protein (GFP). The gene encoding an envelope protein (EnvA) from the avian sarcoma and leukosis virus (ASLV-A) was also inserted into the construct. Target cells in slices of brain from newborn rats were transfected with three genes - the gene encoding an avian receptor protein called TVA (an interaction between EnvA and the TVA receptor occurs during ASLV-A infection of an avian cell), the gene encoding the glycoprotein normally used by the rabies virus to spread from one cell to another, and a gene encoding red fluorescent protein.

Thus, when the construct was introduced into the culture dishes containing the brain slices, it infected only the target cells - those expressing the TVA receptor, the EnvA protein and the red fluorescent protein. Infection with the modified virus caused them to emit a green fluorescence as well. Because the target cells contained the glycoprotein that was deleted from the rabies virus, the construct could spread from the target cells to those connected directly to them by synapses. And, because the modified virus contained GFP, the cells to which it spread began to emit a green fluorescence. But once inside these cells, the viral particles remained stranded there, because only the targeted cells, and not those to which the particles spread, contained the glycoprotein required for them to spread further.

Target cells are labelled red, target cells successfully transfected with the viral construct are labelled red/green and marked with a dashed line, and cells which form synapses with the latter, into which the construct has spread, are labelled green. (From Wickersham, et al, 2007; scale bar = 200 μm)

Upon examination, the brain slices were seen to contain large clusters of green fluorescent neurons surrounding individual red/ green fluorescent cells. This strongly suggested that the modified viruses had spread from the cells into which they had been transfected to cells connected to them. The researchers then used the patch-clamp technique to determine the specificity of this tracing, which was presumed to be transsynaptic. Microelectrodes were inserted into the presumed target cells, and the cells into which the virus was believed to have spread. A solution of positive ions was injected into the cells emitting green fluorescence, causing them to generate action potentials. Consequently, action potentials were recorded in the cells emitting red fluorescence, revealing that the tracer viruses had travelled retrogradely (backwards) and spread from the targeted cells to those connected to them presynaptically (that is, those which send action potentials to them).

This is the first time a transsynaptic tracer has been used to label all the cells connected to a target cell. But it remains unclear whether the viral particles spread to all the neurons which form synapses with the targeted cells. Given more time, the technique may have revealed even more connections that were not made apparent in these experiments. This, and similar techniques being developed by others, will enable researchers to gain a better understanding of neural circuitry. But even accurate tracing methods also have their limitations. Some brain circuits are so complex that one could not hope to accurately trace all the connections within them. In the cerebellum, for example, Purkinje cells form synapses with hundreds of thousands (or perhaps up to a million) parallel fibres. Attempting to visualize the connections of a Purkinje cell, even with the most accurate transsynaptic tracer, would result in an incomprehensible image consisting of a large green blur.

Reference:

Wickersham, I. R., et al. (2007). Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53: 639-647. [Abstract]

This model of nerve cell function has served neuroscientists well for over a hundred years: anatomists and histologists determined the fine structure of the neuron in the latter half of the nineteenth century, and Sir Charles Sherrington coined the term "synapse" in 1897. The model is generally accepted because it provides an explanation of how a brain consisting hundreds of billions of cells, all of which produce action potentials in basically the same way, can work: information is encoded in the patterns of action potentials, and the connectivity of cells is what determines the specificity of neuronal signalling. Thus, action potenitals generated in one cell population will encode different information from signals produced by other populations of cells, because each population projects to a different target region, and therefore release transmitters only onto those target cells.

Recently, researchers have obtained evidence that neurotransmitters can be released from sites other than the presynaptic membrane. For example, at the terminals of climbing fibres, which originate in the inferior olivary nucleus and project to the cerebellar cortex, glutamate is released at sites that do not face the synapse. However, this and similar findings are still consistent with the view that neurotransmitter release occurs at nerve terminals. But now, two papers, published back-to-back in the March issue of Nature Neuroscience, provide evidence that neurotransmitters can be released along the length of axons, suggesting that the classical model of how neurons function may be inadequate.

Both papers describe experiments performed on rodents, and both show that axons in the corpus callosum are capable of releasing the neurotransmitter glutamate, to which adjacent glial cells are responsive. The corpus callosum is the bundle of approximately 100 million axons which connects the two hemispheres of the brain and enables them to communicate with each other. It consists almost entirely of white matter - it contains axons of neurons projecting from one side of the brain to the other, but is devoid of cell bodies or dendrites, and therefore is not a part of the brain where one would expect neurotransmission to take place.

Dirk Dietrich and his colleagues, from the Experimental Neurophysiology Laboratory at the University of Bonn's Department of Neurosurgery, prepared slices of tissue containing the corpus callosum from juvenile rats. At 8-16 days of age, the rat corpus callosum contains large numbers of oligodendrocyte precursor cells (OPCs). Mature oligodendrocytes are the cells which ensheath axons with a fatty protein called meylin; this insulates the fibres and therefore increases the velocity at which they conduct nervous impulses.

Because OPCs are known to express a variety of voltage-gated ion channels, Deitrich's team used the patch clamp technique to record the electrical activity of OPCs. When adjacent corpus callosum axons were stimulated, inward currents were recorded from the OPCs. These currents were recorded from every OPC into which the microelectrodes were inserted, and were always dependent upon stimulation of the adjacent axon. The researchers then used glutamate receptor antagonists to show that the currents are mediated by a type of glutamate receptor called the AMPA receptor; these receptors mediate fast synaptic transmission in the central nervous system. It was also found that OPCs are sensitive to lower concentrations of glutamate than are postsynaptic neurons, and that axons of the rat optic nerve are also capable of releasing glutamate. As in nerve terminals, glutamate is released from both corpus callosum and optic nerve axons in response to local increases in the concentration of calcium ions (calcium "microdomains") in the areas around calcium channels in the axonal membrane.

The molecules involved in synaptic transmission - neurotansmitters and the constituents of synaptic vesicles - are synthesized in the cell body and transported along the axon to the nerve terminal, and, because there is a constant turnover of these proteins, the components of neurotransmission are always present along the length of axons. It is, therefore, possible that the currents recorded in the OPCs were generated by leakage of these components from the axonal transport apparatus. To rule out this possibility, OPCs were continuously stimulated for periods of several seconds. The cells continued to release glutamate, at an estimated 8 times per second, showing that the release was not due to the sporadic leakage of neurotransmitters being transported along the axon.

Electron microscopy was then used to examine the callosal axons. This revealed the presence of synaptic vesicle-like structures in the axons; it showed that at least some of these vesicles were docked at the axonal membrane; and that vesicles vesicles are recycled quickly after use. Thus it appears that the release of neurotransmitters from axons in the corpus callosum occurs in the same way as at classical synapses - by exocytosis. The vesicles in the axons cause small protrusions in the membrane, which form invaginations in the OPCs. However, although the structures in the axon and the OPCs at which transmission occurs are closely apposed to each other, the area between OPCs and axons, into which the neurotransmitter is released, is wider than the cleft of classical synapses. It has an irregular width, and, unlike well-characterized synapses, lacks an extracellular matrix.

Electron micrograph showing a biocytin-laballed oligodendrocyte precursor cell (Bio) and an adjacent callosal axon containing synaptic vesicle-like structures. The region of the axon at which the vesicles are docked causes an invagination of the oligodendrocyte precursor cell membrane. (Scale bar = 200nm; from Kukley, et al, 2007.)

Deitrich's team examined this surprising phenomenon only in juvenile mice. The other paper, by Ziskin et al, describes similar experiments performed in mice, but also shows that axo-glial neurotransmission occurs in the corpus callosum of adult animals. Both papers therefore provide strong evidence that neurotransmitter release is not restricted to nerve terminals, but can also occur at discrete regions along the entire length of the axon. This neuron-glial signalling may provide a means of controlling oligodendrocyte differentitation during neural development. It remains to be seen whether or not this type of signalling takes place in other brain regions.

These findings show that our understanding of basic neuronal function is far from complete, and add yet another level of complexity to information processing in the brain. They also have potential medical applications, as excess release of glutamate can be damaging to nerve cells. This excitotoxicity has been implicated in a wide variety of conditions, including autism and epilepsy, as well as neurodegenerative diseases such as Alzheimer's and Parkinson's. The findings may therefore open up new avenues for the development of treatments for some of these conditions, based on drugs that target glutamate release in the white matter.

References:

Kukley, M. et al. (2007). Vesicular glutamate release from axons in white matter. Nat. Neurosci. 10: 311 - 320. [Abstract]

Ziskin, J. L., et al. (2007). Vesicular release of glutamate from unmyelinated axons in white matter. Nat. Neurosci. 10: 321 - 330. [Abstract]

NFTs were first described by Alois Alzheimer, after his post-mortem examination of the brain of Auguste Deter, the index case of the disease which bears his name. They are found in the pyramidal cells of the neocortex, particularly in temporal lobe structures such as the hippocampus and amygdala, where they appear as flame-shaped structures which fill the cell body and extend into the apical dendrite (left). Tau is normally unfolded and soluble; in Alzheimer's, and related conditions, hyperphosphorylation causes the formation of a tight hairpin loop in the protein, which makes it insoluble. Electron microscopy reveals that, within cortical neurons, NFTs consist of tau fibrils with a diameter of 10 nanometres, coiled around each other to form paired helical structures.

The role of amyloid plaques and NFTs in Alzheimer's disease pathogenesis is not well understood, although the latter are more closely correlated than the former with neuronal dysfunction and the severity of dementia. Recently, researchers have obtained evidence that this protein aggregation may be due to impaired protein degradation, leading to a failure to remove abnormally folded proteins. Also implicated in this process are molecular chaperones, which are involved in recognizing proteins with an abnormal configuration and refolding them; earlier this year, for example, heat shock protein (HSP) 90, a chaperone involved in protein folding and degradation, was implicated in prion diseases.

Leonard Petrucelli and his colleagues at the Mayo Clinic College of Medicine provide more evidence of the role of molecular chaperones in neurodegenerative processes. The findings, which show that inhibition of HSP90 by a small molecule called EC102 leads to increased degradation of toxic p-tau aggregations, are published in advance on the website of the Journal of Clinical Investigation.

Previous work by Petrucelli's team showed that small molecule inhibitors dramatically increase the degradation of p-tau. It was also known that p-tau aggregation increases with loss of function of the carboxy terminus of HSP70-interacting protein (CHIP). To determine the role of the CHIP protein in protein degradation, the researchers performed experiments on cultured HeLa cells overexpressing human tau protein. Using small inhibitory RNAs (siRNAs), expression of the CHIP gene was blocked; as a result, tau protein accumulated in the cells. Conversely, antibody staining showed that, when HSP90 expression was blocked, the number of CHIP-tau complexes increased.

A strain of transgenic mice overexpressing the mutated form of human tau protein was then created. Intraperitoneal injection of EC102 led to increased degradation of the abnormal protein, and reduction in p-tau aggregates by approximately 50%. Finally, the brains of deceased Alzheimer's patients were compared with the brains of controls who had died of other causes. HSP90 was found to be tightly bound to p-tau aggregates in the affected regions of the Alzheimer's patients, but not in the controls.

Taken together, the data suggest that CHIP mediates the interactions of molecular chaperones with their "client" proteins, such as p-tau. It appears that the chaperones compete in binding with abnormal tau protein, and that binding of HSP90 to p-tau aggregates prevents CHIP from recruiting other chaperones that would induce degradation of the aggregates. Inhibition of HSP90 reduces this competition, leading to targeted degradation of p-tau.

The findings also support the idea that there is an age-related loss of molecular chaperone function, which may be exaserbated in Alzheimer's. Because EC102 can pass through the blood-brain barrier, it, and related compounds, may prove to be effective treatments for Alzheimer's and other "tauopathies" (conditions in which there is aggregation and deposition of tau protein), such as Pick's disease, Parkinson's-associated frontotemporal dementia; supranuclear palsy and corticobasal degeneration;

Reference:

Dickey, C. A. (2007). The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins. J. Clin. Invest. DOI: 10.1172/JCI129715. [Full text]

Related:

• Study shows how prion diseases sow their seeds
• 100 years of Alzheimer's Disease
  

One of the central dogmas of neuroscience was that the brains of adult mammals cannot generate new nerve cells. But about 10 years ago, this changed, when it was discovered that in the hippocampi of adult rodents there are stem cells which are capable of generating new neurons. It has also been known for some years that, in rodents at least, neurogenesis also produces new cells that migrate to the olfactory bulb. Although a study published in 1998 provided some evidence that neurogenesis takes place in the adult human hippocampus, the idea that neurogenesis takes place in adult humans remained contentious.

Now, an advance online publication on the website of the journal Science provides evidence that neurogenesis may also occur in the olfactory bulb of the human brain:

The rostral migratory stream (RMS) is the main pathway by which newly born subventricular zone (SVZ) cells reach the olfactory bulb in rodents. However, the RMS in the adult human brain has been elusive. Here we demonstrate the presence of a human RMS, which is unexpectedly organized around a lateral ventricular extension reaching the olfactory bulb (OB), and illustrate the neuroblasts in it. The RMS ensheathing the lateral olfactory ventricular extension, as seen by MRI, cell specific markers and electron microscopy, contains progenitor cells with migratory characteristics and cells which incorporate BrdU and become mature neurons in the OB.

In rats and mice, a structure called the rostral extension connects the lateral ventricles and the olfactory bulb. This tube-like structure, which is filled with cerebrospinal fluid, provides a pathway for neurons generated in the subventricular zone (a proliferative tissue lining the cerebral ventricles) to migrate to the olfactory bulb, which contains neurons that bind to odorant receptors and produce nervous impulses related to smell. The rostral migratory stream is the equivalent of the rostral extension in rodents, and was not known to exist in humans before this study.

Maurice Curtis and his colleagues examined the brains of deceased cancer patients who had previously been injected with bromo- deoxyuridine (BrdU), a chemical which is incorporated into newly-synthesized DNA, and which is therefore used by oncologists to visualize and monitor the growth of tumours. To their surprise, they found BrdU-positive cells in the olfactory bulbs of the patients' brains, suggesting that it contained newly-generated neurons. Curtis's team then used antibody staining to show that the neuroblasts begin to differentiate into olfactory neurons while migrating through the rostral migratory stream. Upon arriving at the bulb, the cells continued to differentiate, forming mature olfactory neurons. Using electron microscopy, they also showed that this 'tube' is 3.5 mm long and 1.5 mm in diameter.

Because the cancer patients whose brains were examined were aged between 38-70 years of age, the findings suggest that neuro- genesis may occur throughout the duration of the human lifespan. The function of these newly-generated cells is unclear, but they may be involved in recognizing and remembering new smells in the later years of life.

The researchers also have preliminary unpublished data that the stem cells not only migrate to the olfactory bulb, but also leave the RMS and migrate into the basal ganglia and cerebral cortex. This is significant, because parts of the basal ganglia degenerate in movement disorders such as Parkinson's Disease, and specific regions of the cortex degenerate in Alzheimer's. The possibility that stem cells enter these regions from the RMS could therefore provide a means for developing new treatments for neurodegenerative diseases.

Update: Evidence of neurogenesis in the adult human olfactory bulb was obtained by Bedard and Parent in 2004.

Update 2: The figure on the right (from Gray's Anatomy) shows the simple cellular structure of the distal tip of the human olfactory bulb. The olfactory cell is a primary sensory neuron that contains olfactory receptors, which are activated by the binding of odorant molecules. In the glomerulus, olfactory cells synapse with the secondary cells, which form the olfactory nerve (cranial nerve I).

The olfactory epithelium is a neuroepthelium - a single layer of cells, in which the cell bodies of the olfactory cells nestle in between specialized epithelial cells. This single cell layer forms the roof of the nasal cavity, so one side of layer is exposed to the open air. It is therefore easily damaged by, for example, inhalation of toxic fumes; olfactory epithelial cells retain their capacity for neurogenesis into adulthood, so that damaged cells can be replaced after minor injuries. The other cells in the olfactory bulb are similarly exposed to air, but to a lesser extent. This is the likely reason that the tissue has retained its regenerative capacity.

References:

Curtis, M. A. et al. (2007). Human neuroblasts migrate to the olfactory bulb via a lateral ventricular extension. Science (Advance online publication) DOI: 10.1126/science.1136281

Gritti, A., et al. (2002). Multipotent neural stem cells reside in the rostral extension and olfactory bulb of adult rodents. J. Neurosci. 22: 437-445.

Bedard, A. & Parent, A. (2004). Evidence of newly generated neurons in the human olfactory bulb. Brain Res. Dev. Brain Res. 151: 159-168.

Costanzo, R. M. (2005). Regeneration and rewiring of the olfactory bulb. Chem. Senses Suppl 1: i133-i134.

The footage below was obtained between 240-940 metres deep, near Ogasawara Islands in the North Pacific, and shows the squid producing bioluminescent displays as it circles torch lights attached to a bait rig. As the squid approaches the bait, it spreads its arms widely and emits short bright flashes of bioluminescence, lasting about 1 second. As well as dazzling and disorienting prey, these displays may also serve to illuminate the squid's deep sea environment, thus enabling it to measure the distance between itself and its prey. When the lights from the bait rig were switched on, the squid produced longer bioluminescent flashes, lasting about 5 seconds. The researchers believe that these may be courtship displays.

T. danae can reach more than 2 metres in length and is thought to be one of the largest mesopelagic (or mid-depth) cephalopods found in tropical and sub-tropical oceans. This is the first time that a giant squid has been observed using bioluminescence when hunting. Although large, the giant squid is an active predator that is far from sluggish in its movements - it can reach speeds of up to 2.5 metres per second, swims both forwards and backwards, and can use its large triangular fins to change direction quickly.

A subtler, but perhaps more dazzling bioluminescent display is that of the vampire squid Vampyroteuthis infernalis:

There are 100 known genera of squid and cuttlefish, 63 of which produce bioluminescence. Cephalopods possess light-producing organs called photophores, which can be arranged in simple clusters or in complex organs containing lenses, reflectors and light filters. In some cephalopods, light is produced by symbiotic bacteria which are housed and cultured within pockets in the skin; other species produce the light themselves.

Squid also use bioluminescence to avoid being detected by predators. In a process called countershading, they turn on downward- facing photophores when illuminated from above; when the intensity of the bioluminescence matches that of the overhead illumination, the squid becomes invisible.

Bioluminescence is produced when pigment molecules called luciferins are oxidized (i.e. have electrons removed from them). This reaction is carried out by enzymes called luciferases, which are believed to have evolved from oxygenases. The function of luciferases and oxygenases is to transfer electrons from one molecule to another, and they are therefore involved in neutralizing free radicals (compounds which contain unpaired electrons, and which are damaging to tissues). In the deep sea environment there is little light, and organisms are therefore not exposed to free radicals produced by direct sunlight as are organisms which live nearer the surface. Neutralization of free radicals is therefore not crucial in deep waters, and this is where luciferases evolved their light-producing functions.

The photophores found in T. danae measure up to 5 cm in diameter and are the largest found in the animal kingdom. They are equipped with black "eyelids" which can open and shut rapidly, so that the emitted light appears to flash on and off.

Update (22nd February, 2007). From MSNBC:

WELLINGTON, New Zealand - A fishing crew has caught a colossal squid that could weigh a half-ton and prove to be the biggest specimen ever landed, a fisheries official said Thursday. The squid, weighing an estimated 990 pounds and about 39 feet long, took two hours to land in Antarctic waters, New Zealand Fisheries Minister Jim Anderton said. [snip] "I can assure you that this is going to draw phenomenal interest. It is truly amazing," said Steve O'Shea, a squid expert at the Auckland University of Technology. If calamari rings were made from the squid they would be the size of tractor tires, he added.

References:

Kubodera, T. et al. (2007). Observations of wild hunting behaviour and bioluminescence of a large deep sea, eight-armed squid, Taningia danae. Proc. R. Soc. B. DOI: 10.1098/rspb.2006.0236.

Kubodera, T. & Mori, K. (2005). First-ever observations of a live giant squid in the wild. Proc. R. Soc. B. 272: 2583–2586.

Related:

• Super-reflective protein found in octopus skin
• Researchers reveal the squid's hidden messages
  

To date, 158 British people have died from vCJD, and 7 more are known to be infected. And research performed last year by John Collinge, the senior author of the current study, suggests that human prion diseases may have an incubation period of up to 50 years, and that an epidemic of vCJD may lie ahead. It is widely believed that the transmission of prion diseases to humans occurs by the consumption of infected meat, which contains an abnormal version of a protein found in all nerve cells.

The full name of the prion diseases - spongiform encephalopathies - means "sponge-like brain diseases", and describes the appearance of infected nervous tissue, which becomes dotted with small "holes" as the disease progresses. These holes make the tissue resemble a sponge, and eventually interfere with the ability of nerve cells to conduct electrical impulses. This is followed by damage to synapses and death of affected nerve cells. But the symptoms of prion diseases, which include loss of motivation, cognitive impairment, and unsteady gait, are exhibited before a diagnosis can be made, and before any cell death occurs.

The new work, which was led by Giovanna Mallucci and has just been published in the journal Neuron, involved the creation of a strain of transgenic mice. This involved making a genetic construct consisting of a copy of the prion gene containing DNA sequences that are recognized by an enzyme which snips DNA and rearranges it by a process called recombination. The animals were infected with abnormal prion protein at around one week of age. Soon after infection, all the mice displayed the cognitive and behavioural symptoms of prion infection. They exhibited impaired recognition of familiar objects, and burrowing behaviour was reduced due to a lack of motivation. These symptoms were correlated with damage to the dorsal region of the hippocampus; immunohistochemical staining showed reduced levels of a nerve terminal protein called synaptophysin 1, indicating that synapses were damaged, and electrophysiological recordings confirmed that there was also a decline in synaptic function in that region of the brain.

When the mice reached 10 weeks of age, the gene encoding the DNA recombination enzyme was activated in one group of mice, so that the prion gene was disrupted and synthesis of the prion protein ceased. In the other group, the prion gene was kept intact, and the cells continued to synthesize the protein. It was found that, in the mice that had stopped expressing the prion gene, depletion of the protein led to a reversal of the disease symptoms and pathologies. The memory of infected animals, as measured by their performance on the object recognition task, was restored, and they performed as well as uninfected control mice. The improved performance paralleled a reversal of pathological hallmarks of prion diseases: antibody staining showed that synaptophysin 1 levels in the dorsal hippocampus had returned to normal, and electrophysiological recordings showed a recovery of synaptic function. On the other hand, in the group of mice that continued to express the prion gene, the symptoms progressed, leading ultimately to the animals' death.

This is the first study to show that the symptoms and pathology of prion diseases are reversible, and the findings may have implications for other neurodegenerative diseases, particularly Alzheimer's. However, the reversal of symptoms and pathology observed in this study was only possible because the depletion of prion protein was induced in the very early stages of infection. The recovery of function may only have occurred because the mice were so young; in older animals, in which there is less brain plasticity, this recovery may not have occurred. Mallucci and her colleagues therefore stress that any possible treatment for vCJD - based on drugs which block prion protein function - could take years to develop, and would depend on a blood test that can detect prion diseases in the early stages, before any irreversible damage to the brain has occurred. Furthermore, because the prion protein is a normal constituent of nerve cells, the long-term effects of preventing its synthesis are unclear.

Reference:

Mallucci, G. R. et al (2007). Targeting Cellular Prion Protein Reverses Early Cognitive Deficits and Neurophysiological Dysfunction in Prion-Infected Mice. Neuron 53: 325-335.

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New research led by Tricia Serio, an assistant professor of molecular cell biology and biochemistry at Brown University in Providence, Rhode Island, has identified a protein which appears to be crucial for self-replication of the abnormal prion protein and for propagation of the "seed" in the budding yeast Saccharomyces cerevisiae. The work is published today in the online open-access journal PLoS Biology.

The protein implicated is heat shock protein (HSP) 104. HSPs are a large family of proteins present in all cells, which have previously been implicated in prion disease pathogenesis. HSP levels increase in response to exposure of the cells to elevated temperatures. They also act as molecular chaperones, assisting newly-formed proteins to fold up into their proper three-dimensional conformation, and preventing them from accumulating in clumps. It is this latter role which appears crucial for the inheritance of abnormally folded prion protein during division of yeast cells.

Serio's team constructed a mutated form of the HSP104 gene, and a fusion of the gene encoding green fluorescent protein (GFP) and the gene encoding the yeast prion protein Sup35 . Both constructs were then inserted into yeast cells; because it was fused to GFP, the Sup35 molecules emitted a green fluorescence, enabling them to be visualized easily.

Examination of the yeast cells containing the mutated, non-functional HSP104 gene showed that Sup35 still formed toxic aggregates. However, it was found that there was a dramatic reduction in the motility of  the aggregates within the cytoplasm. They remained clumped together, fragmenting at a far slower rate than they normally would, such that, during cell division, the abnormal prion was not transmitted to daughter cells. This also occurred when HSP104 was inhibited by addition of a chemical called guanidine hydrochloride.

The authors conclude that HSP104 is crucial for the seeding mechanism. They propose a model whereby disassembly of prion protein clumps is dependent on HSP104 function. In the figure below, which illustrates the model, the normal soluble form of Sup35 molecule is shown in green; the abnormal form of Sup35, which is insoluble and prone to aggregation, is shown in black; and HSP104 is shown as a barrel-shaped structure.

In 2005, the same team found that the abnormally folded Sup35 can induce normal Sup35 molecules to adopt the toxic conformation within a single cycle of cell division. It is because of this ability to rapidly seed the formation of new aggregates that a prion disease infection spreads quickly through the brain of an infected organism. Thus, the new findings could lead to the development of drugs which prevent or slow the spread of infection throughout the brain. Furthermore, it was recently discovered that amyloid-beta protein, which is associated with Alzheimer's Disease, can also be propagated by the same seeding mechanism; the findings may, therefore, also lead to treatments that slow the progression of Alzheimer's, and other neurodegenerative diseases in which there is an intracellular accumulation of toxic, insoluble proteins.

References:

Satpute-Krishnan, P. et al. (2007). HSP104-dependent remodelling of prion complexes mediates protein-only inheritance. PLoS Biology 5: e24 DOI:10.1371/journal.pbio.0050024.

Satpute-Krishnan, P. & Serio, T. R. (2005). Prion protein remodelling confers an immediate phenotypic switch. Nature 437: 262-265.

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