(Image caption: During development, nerve cells (shown in blue, green, violet and yellow) extend their axons to target leg muscles. If the EphA4 receptors of the growing nerve cells no longer encounter freely accessible ephrins, the axons of many nerve cells (violet) are no longer able to find their partner cells. Credit: © MPI of Neurobiology / Gatto)

Neurons in a forest of signposts

Our ability to move relies on the correct formation of connections between different nerve cells and between nerve and muscle cells. Growing axons of nerve cells are guided to their targets by signposts expressed on the surface of other cells. Very prominent are “do not enter” signs that push axons away. Cell culture studies suggest that protein-cutting enzymes (proteases) remove these signs as soon as they are recognized by the growing axons. In this way, the “bond of recognition” between the axon and the sign is quickly broken, and the axons are more easily guided in a new direction. Scientists from the Max Planck Institute of Neurobiology in Martinsried and the Institut de Recherches Cliniques de Montréal have now shown that proteases indeed control the navigation of growing axons. However, contrary to the current belief, they do so by regulating the number of existing signs. Without proteases, the signposts would be masked and the axons would grow in the wrong direction. These findings clarified how cells form connections during development and may also improve our understanding of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

The human brain consists of about 100 billion nerve cells. During embryonic development each of these cells connects with other cells by means of a long extension, known as axon. Some axons need to navigate long distances through the body to find their correct targets, for example from the spinal cord down to the foot. Only if all these connections are correctly established we can perform basic and fine-tuned movements, such as walking or playing the piano.

It is therefore essential that each nerve cell finds its correct target. But how does an axon navigate and find the appropriate partner cells among billions of other possible targets? “We have now identified a few dozen guidance molecules and their receptors that help axons orient themselves,” says Rüdiger Klein, Director at the Max Planck Institute of Neurobiology. “However, these few receptor-guidance molecule pairs need to control a very large number of navigational decisions. Therefore, there must be some mechanisms to amplify and modulate the effects of these protein pairs”.

Cutting for speed?

Over the last decade, Rüdiger Klein and his team have been studying how nerve cells find their way during development. They are focusing on “do not enter” signs, e.g. ephrin guidance molecules and their Eph receptors. Ephrins and Eph receptors, being present on almost all cell surfaces: on axons as well as on cells in the surrounding tissues, help the growing axons to explore their surroundings and locate their partner cells.

As an axon travels through the body, it docks again and again to other cells via the ephrin/Eph system. This triggers cellular processes, in one or both cells, that eventually cause the connection to be severed and the cells to repel each other, preventing the axon to grow in the wrong direction. It has been hypothesised that this cellular repulsion is accelerated by proteases. Proteases are enzymes that cut Eph receptors and/or ephrins, thus by severing the Eph/ephrin bond between two opposing cells they might expedite the repulsion process. “In this way, proteases could contribute to changes in the guidance process – but this has not yet been experimentally proven.” says Rüdiger Klein.

Not faster, but better

To address this question, the neurobiologists studied how proteases affect the rate of cellular repulsion controlled by EphA4 receptors and ephrins. “Although the experiments in cell culture initially appeared to confirm the theory, we discovered something quite different in living organisms,” states Rüdiger Klein. Contrary to expectations, cellular repulsion proceeded with undiminished accuracy in animals whose axons expressed EphA4 receptors resistant to protease severing. On the other hand, in animals whose axons and muscles expressed EphA4 receptors resistant to protease cutting many axons grew in the wrong direction. Because no cutting occurred, more and more functional EphA4 receptors accumulated on cell surfaces of the leg tissues. This accumulation caused EphA4 receptors to bind to the ephrins on the same cell surface, a phenomena termed as “masking”. Consequently, the ephrins could no longer act as “do not enter” signs for the growing axons. Thus, axons, being no longer repelled, are misguided in a “no entry zone” and are unable to find their correct targets.

These results show that the cleavage of Eph receptors by proteases does not, as expected, accelerate the repulsion reaction. Instead, it regulates the number of functioning receptors and indirectly the number of available ephrins on cells, where they serve as navigational aids. If the balance is disrupted, growing axons are misdirected.

This is an important finding, as EphA4 receptors perform essential functions during the development of neural networks in the brain and in the spinal cord. They are also involved in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In the absence of EphA4 receptors, ALS manifests itself later and develops more slowly in a number of animal models. “It’s possible that the number of EphA4 receptors is kept low by the regulatory activity of proteases,” Rüdiger Klein reflects. “This could provide a way to exert a positive influence on the course of ALS.”

(Image caption: Neurons with the Unc5-receptor send their axons in a cell culture in all directions. The processes avoid the parallel orientated stripes containing the FLRT3-protein (red). Credit: ©Seiradake et al, Neuron 2014)

Navigation for nerve cells

During brain development, the precursors of nerve cells sometimes have to migrate long distances from their place of origin to their destination. In this process, proteins, such as FLRTs (pronounced “flirts”), act as guide molecules. Researchers at the Max Planck Institute of Neurobiology in Martinsried, together with colleagues at the Universities of Oxford and Frankfurt have now discovered that FLRT proteins on the surface of progenitor cells can induce repellent and attractant signals depending on its binding partner. The scientists used X-ray crystallography to reveal the structural bases for both FLRT-mediated adhesion and repulsion. They applied this knowledge to elucidate how these opposed signals control cellular migration. Which signal predominates depends on the particular type of cell migration. The results further show that FLRTs also exert attractant and repellent effects in the walls of blood vessels and therefore control the development of other tissue types as well.

Pyramidal cells are the central nerve cells in the cerebral cortex. During embryonic development, the precursors of pyramidal cells follow the paths of glial cell axons to migrate from their original location to the surface of the cerebral cortex. As soon as they reach their intended layer, they develop into mature pyramidal cells and interlink to form a functional network. Pyramidal cells also spread to a limited extent within these layers, though the importance of such tangential migration is still poorly understood.

This migration of precursor pyramidal cells is controlled by FLRTs (fibronectin-leucine-rich transmembrane proteins) located on the cell surface. According to the researchers at the Max Planck Institute in Martinsried, FLRTs and the Unc5 receptor form a group of guidance proteins with opposing effects on cell migration. On one hand, they act as a repellent. This is the case when a FLRT molecule binds to an Unc5 receptor on the surface of a progenitor cell. “In this way, as the precursor cell migrates radially, it receives a signal to continue migrating at an adjusted speed to not move prematurely into outer layers,” explains Rüdiger Klein from the Max Planck Institute of Neurobiology.

However, if two identical FLRT molecules bind to each other, this triggers an adhesive signal. The scientists’ results show that pyramidal cells are guided in this manner as they spread tangentially, without affecting their ability to find their target layer. Thus, there are proteins with attractant and repellent effects located on the surface of precursor pyramidal cells. “By integrating these opposing signals, cells can navigate through brain tissue. During radial migration FLRTs induce repulsion; during tangential dispersion FLRT attraction dominates,” says Klein.

In their study the scientists also investigated whether the mechanisms of FLRT adhesion and repulsion are present in other cell types. Their findings show that cells in the walls of blood vessels in the retina and the umbilical cord are also controlled by a combination of attractant and repellent signals modulated by FLRT and Unc5 proteins.

Mechanism that repairs brain after stroke discovered

A stroke is caused by a blood clot blocking a blood vessel in the brain, which leads to an interruption of blood flow and therefore a shortage of oxygen. Many nerve cells die, resulting in motor, sensory and cognitive problems.

The researchers have shown that following an induced stroke in mice, support cells, so-called astrocytes, start to form nerve cells in the injured part of the brain. Using genetic methods to map the fate of the cells, the scientists could demonstrate that astrocytes in this area formed immature nerve cells, which then developed into mature nerve cells.

”This is the first time that astrocytes have been shown to have the capacity to start a process that leads to the generation of new nerve cells after a stroke”, says Zaal Kokaia, Professor of Experimental Medical Research at Lund University.

The scientists could also identify the signalling mechanism that regulates the conversion of the astrocytes to nerve cells. In a healthy brain, this signalling mechanism is active and inhibits the conversion, and, consequently, the astrocytes do not generate nerve cells. Following a stroke, the signalling mechanism is suppressed and astrocytes can start the process of generating new cells.

”Interestingly, even when we blocked the signalling mechanism in mice not subjected to a stroke, the astrocytes formed new nerve cells”, says Zaal Kokaia.

“This indicates that it is not only a stroke that can activate the latent process in astrocytes. Therefore, the mechanism is a potentially useful target for the production of new nerve cells, when replacing dead cells following other brain diseases or damage.”

The new nerve cells were found to form specialized contacts with other cells. It remains to be shown whether the nerve cells are functional and to what extent they contribute to the spontaneous recovery that is observed in a majority of experimental animals and patients after a stroke.

A decade ago, Kokaia’s and Lindvall’s research group was the first to show that stroke leads to the formation of new nerve cells from the adult brain’s own neural stem cells. The new findings further underscore that when the adult brain suffers a major blow such as a stroke, it makes a strong effort to repair itself using a variety of mechanisms.

The major advancement with the new study is that it demonstrates for the first time that self-repair in the adult brain involves astrocytes entering a process by which they change their identity to nerve cells.

”One of the major tasks now is to explore whether astrocytes are also converted to neurons in the human brain following damage or disease. Interestingly, it is known that in the healthy human brain, new nerve cells are formed in the striatum. The new data raise the possibility that some of these nerve cells derive from local astrocytes. If the new mechanism also operates in the human brain and can be potentiated, this could become of clinical importance not only for stroke patients, but also for replacing neurons which have died, thus restoring function in patients with other disorders such as Parkinson’s disease and Huntington’s disease”, says Olle Lindvall, Senior Professor of Neurology.

Manipulating memory with light

Just look into the light: not quite, but researchers at the UC Davis Center for Neuroscience and Department of Psychology have used light to erase specific memories in mice, and proved a basic theory of how different parts of the brain work together to retrieve episodic memories.

Optogenetics, pioneered by Karl Diesseroth at Stanford University, is a new technique for manipulating and studying nerve cells using light. The techniques of optogenetics are rapidly becoming the standard method for investigating brain function.

Kazumasa Tanaka, Brian Wiltgen and colleagues at UC Davis applied the technique to test a long-standing idea about memory retrieval. For about 40 years, Wiltgen said, neuroscientists have theorized that retrieving episodic memories — memories about specific places and events — involves coordinated activity between the cerebral cortex and the hippocampus, a small structure deep in the brain.

"The theory is that learning involves processing in the cortex, and the hippocampus reproduces this pattern of activity during retrieval, allowing you to re-experience the event," Wiltgen said. If the hippocampus is damaged, patients can lose decades of memories.

But this model has been difficult to test directly, until the arrival of optogenetics.

Wiltgen and Tanaka used mice genetically modified so that when nerve cells are activated, they both fluoresce green and express a protein that allows the cells to be switched off by light. They were therefore able both to follow exactly which nerve cells in the cortex and hippocampus were activated in learning and memory retrieval, and switch them off with light directed through a fiber-optic cable.

They trained the mice by placing them in a cage where they got a mild electric shock. Normally, mice placed in a new environment will nose around and explore. But when placed in a cage where they have previously received a shock, they freeze in place in a “fear response.”

Tanaka and Wiltgen first showed that they could label the cells involved in learning and demonstrate that they were reactivated during memory recall. Then they were able to switch off the specific nerve cells in the hippocampus, and show that the mice lost their memories of the unpleasant event. They were also able to show that turning off other cells in the hippocampus did not affect retrieval of that memory, and to follow fibers from the hippocampus to specific cells in the cortex.

"The cortex can’t do it alone, it needs input from the hippocampus," Wiltgen said. "This has been a fundamental assumption in our field for a long time and Kazu’s data provides the first direct evidence that it is true."

They could also see how the specific cells in the cortex were connected to the amygdala, a structure in the brain that is involved in emotion and in generating the freezing response.

(Image caption: Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides. Credit: Institute of Molecular Cell Biology)

Vesicles influence the function of nerve cells

Tiny vesicles containing protective substances which they transmit to nerve cells apparently play an important role in the functioning of neurons. As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered, nerve cells can enlist the aid of mini-vesicles of neighboring glial cells to defend themselves against stress and other potentially detrimental factors. These vesicles, called exosomes, appear to stimulate the neurons on various levels: they influence electrical stimulus conduction, biochemical signal transfer, and gene regulation. Exosomes are thus multifunctional signal emitters that can have a significant effect in the brain.

The researchers in Mainz already observed in a previous study that oligodendrocytes release exosomes on exposure to neuronal stimuli. These exosomes are absorbed by the neurons and improve neuronal stress tolerance. Oligodendrocytes are a type of glial cell and they form an insulating myelin sheath around the axons of neurons. The exosomes transport protective proteins such as heat shock proteins, glycolytic enzymes, and enzymes that reduce oxidative stress from one cell type to another, but also transmit genetic information in the form of ribonucleic acids.

"As we have now discovered in cell cultures, exosomes seem to have a whole range of functions," explained Dr. Eva-Maria Krämer-Albers. By means of their transmission activity, the small bubbles that are the vesicles not only promote electrical activity in the nerve cells, but also influence them on the biochemical and gene regulatory level. "The extent of activities of the exosomes is impressive," added Krämer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.

(Image caption: The structure determines the function: AMPA receptors in the nerve cells of the brain are composed of a range of more than 30 different proteins. Source: Bernd Fakler)

Understanding the Components of Memory

Dr. Uwe Schulte, Dr. Jochen Schwenk, Prof. Dr. Bernd Fakler, and their team have elucidated the enormous spatial and temporal dynamics in protein composition of the AMPA-type glutamate receptors, the most important excitatory neurotransmitter receptors in the brain. These receptors are located in the synapses, the contact points between two nerve cells, where they are responsible for the rapid signal transduction and information processing. The results illustrate that the receptors are far more diverse than previously anticipated and pave the way for research into their functions in the various regions of the brain. The biologists published their findings in the journal Neuron.

The researchers have thus opened up the possibility to investigate the properties and functions of the AMPA receptors in the various regions of the brain at the level of their protein components. This is of particular significance as the AMPA receptors and their dynamics are regarded as central elements for memory formation. The researchers succeeded in elucidating the subunit structure of the AMPA receptors in various regions of the brain and even in different groups of distinct nerve cells. It became clear that the receptors exhibit an enormous range of variation in structure and molecular architecture and can evidently be precisely adapted to the function of the nerve cells and brain region in which they are located. In addition, the researchers demonstrated that this diversity in protein composition of the receptors is also exploited during the development of the brain.

In 2012, Fakler’s research team already used novel proteomic technologies to show that AMPA receptors in the brain are assembled from a pool of more than 30 different proteins - whose primary function(s) is are most parts as yet unknown. In fact in another recent study, also published in Neuron, the researchers demonstrated just how significant these unknown components are or can be: They showed that the cornichon protein dictates the time course of the AMPA receptor-mediated synaptic transmission and thus accounts for the difference between various types of nerve cells in the brain.

New learning mechanism for individual nerve cells

“This means a dramatic increase in the brain’s learning capacity. The cells we have studied control the blink reflex, but there are many cells of the same type that control entirely different processes. It is therefore likely that the timing mechanism we have discovered also exists in other parts of the brain”, said Professor of neurophysiology Germund Hesslow.

Professor Hesslow and colleagues Fredrik Johansson and Dan-Anders Jirenhed have used ‘conditioned reflexes’ for the research. The principle comes from the Russian researcher Ivan Pavlov, who, around the turn of the last century, taught dogs to associate a certain sound with food so that they began to drool on hearing the sound.

In the present experiment, the researchers studied animals that learnt to associate a sound with a puff of air in the eye that caused them to blink. If the time between the sound and the puff of air was quarter of a second, the animals blinked after quarter of a second even if the puff of air was removed. If the time was changed to half a second, the animals blinked after half a second, and so on.

The prevalent theories in brain research state that this learnt timing mechanism is a result of strengthening or weakening of the contacts – or synapses – throughout a network of nerve cells. However, using super-thin electrodes, the Lund group have now shown that no networks are needed: one single cell can learn when it is time to react.

The cells which the researchers have studied are called Purkinje cells and are located in the cerebellum. The cerebellum is the part of the brain responsible for posture, balance and movement, and the researchers focused on those cells that control blinking.

This work is basic research, but possible future applications could include rehabilitation following a stroke, which often affects a patient’s movements. The findings could also have a bearing on conditions such as autism, ADHD and language problems, in which the cerebellum is believed to play a part.

“Intelligible speech is dependent on correct timing, so that the pauses between the sounds are right”, explained Germund Hesslow.

The new findings have already attracted attention in the research community: the internationally renowned memory researcher Charles Gallistel came all the way from Rutgers University in the spring to study the group’s work. Work is now continuing to study what transmitter substance and what receptor on the surface of the cell are responsible for the newly discovered timing mechanism.

New findings on how brain handles tactile sensations

The traditional understanding in neuroscience is that tactile sensations from the skin are only assembled to form a complete experience in the cerebral cortex, the most advanced part of the brain. However, this is challenged by new research findings from Lund University in Sweden that suggest both that other levels in the brain play a greater role than previously thought, and that a larger proportion of the brain’s different structures are involved in the perception of touch.

“It was believed that a tactile sensation, such as touching a simple object, only activated a very small part of the cerebral cortex. However, our findings show that a much larger part is probably activated. The assembly of sensations actually starts in the brainstem”, said neuroscience researcher Henrik Jörntell at Lund University.

According to his colleague Fredrik Bengtsson, who also participated in the research, this is the first study to show how complex tactile sensations from the skin are coded at the cellular level in the brain.

“Our findings have given us a new key to understanding how the perception of touch in the skin is processed and communicated to the brain”, he said.

The Lund researchers have worked in collaboration with researchers in Paris to study how individual nerve cells receive information from the skin. They used a ‘haptic interface’, which created controlled sensations of rolling and slipping movements and of contact initiating and ceasing. Movements proved decisive for the perception of touch – something that was not previously technically possible to study.

The findings of the Swedish-French research group have been published in the distinguished journal Neuron. The work is based on animal experiments and is first and foremost basic research, which aims to increase knowledge of the function of the brain. However, there are also possible areas of application.

“Normal hand and arm prostheses do not give any feedback and therefore no sensation of being a ‘real’ hand or arm. However, there are new, advanced prostheses with sensors that can supply information to the amputated arm. Our research could contribute to the further development of such sensors”, said Henrik Jörntell.

The new findings could also have a bearing on psychiatric illness and brain diseases such as stroke and Parkinson’s disease. Detailed knowledge of how the brain and its various parts process information and create a picture of a tactile experience is important to understanding these conditions.

“If we know how a healthy brain operates, we can compare it with the situation in different diseases. Then perhaps we can help patients’ brains to function more normally”, said Henrik Jörntell.

(Image caption: A neuron in which the axon originates at a dendrite. Signals arriving at this dendrites become more efficiently forwarded than signals input elsewhere. Credit: Alexei V. Egorov, 2014)

Communication without detours

Certain nerve cells take a shortcut for the transmission of information: signals are not conducted via the cell`s center, but around it like on a bypass road. The previously unknown nerve cell shape is now presented in the journal “Neuron" by a research team from Heidelberg, Mannheim and Bonn.

Nerve cells communicate by using electrical signals. Via widely ramified cell structures—the  dendrites—, they receive signals from other neurons and then transmit them over a thin cell extension—the axon—to other nerve cells. Axon and dendrites are usually interconnected by the neuron’s cell body. A team of scientists at the Bernstein Center Heidelberg-Mannheim, Heidelberg University, and the University of Bonn has now discovered neurons in which the axon arises directly from one of the dendrites. Similar to taking a bypass road, the signal transmission is thus facilitated within the cell.

“Input signals at this dendrite do not need not be propagated across the cell body,” explains Christian Thome of the Bernstein Center Heidelberg-Mannheim and Heidelberg University, one of the two first authors of the study. For their analyses, the scientists specifically colored the places of origin of axons of so-called pyramidal cells in the hippocampus. This brain region is involved in memory processes. The surprising result: “We found that in more than half of the cells, the axon does not emerge from the cell body, but arises from a lower dendrite,” Thome says.

The researchers then studied the effect of signals received at this special dendrite. For this purpose, they injected a certain form of the neural transmitter substance glutamate into the brain tissue of mice that can be activated by light pulses. A high-resolution microscope allowed the neuroscientists to direct the light beam directly to a specific dendrite. By the subsequent activation of the messenger substance, they simulated an exciting input signal.

“Our measurements indicate that dendrites that are directly connected to the axon, actively propagate even small input stimuli and activate the neuron,” says second first author Tony Kelly, a member of the Sonderforschungsbereich (SFB) 1089 at the University of Bonn. A computer simulation of the scientists predicts that this effect is particularly pronounced when the information flow from other dendrites to the axon is suppressed by inhibitory input signals at the cell body.

“That way, information transmitted by this special dendrite influences the behavior of the nerve cell more than input from any other dendrite,” Kelly says. In a future step, the researchers attempt to figure out which biological function is actually strengthened through the specific dendrite—and what therefore might be the reason for the unusual shape of these neurons.

New Molecular Target is Key to Enhanced Brain Plasticity

As Alzheimer’s disease progresses, it kills brain cells mainly in the hippocampus and cortex, leading to impairments in “neuroplasticity,” the mechanism that affects learning, memory, and thinking. Targeting these areas of the brain, scientists hope to stop or slow the decline in brain plasticity, providing a novel way to treat Alzheimer’s. Groundbreaking new research has discovered a new way to preserve the flexibility and resilience of the brain.

The study, led by Tel Aviv University’s Prof. Illana Gozes and published in Molecular Psychiatry, reveals a nerve cell protective molecular target that is essential for brain plasticity. According to Prof. Gozes, “This discovery offers the world a new target for drug design and an understanding of mechanisms of cognitive enhancement.”

Prof. Gozes is the incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors and director of the Adams Super Center for Brain Studies at the Sackler Faculty of Medicine and a member of TAU’s Sagol School of Neuroscience. Also contributing to the study were Dr. Saar Oz, Oxana Kapitansky, Yanina Ivashco-Pachima, Anna Malishkevich, Dr. Joel Hirsch, Dr. Rina Rosin-Arbersfeld, and their students, all from TAU. TAU staff scientists Dr. Eliezer Gildai and Dr. Leonid Mittelman provided the state-of-the-art molecular cloning and cellular protein imaging necessary for the study.

Building on past breakthroughs

The new finding is based on Prof. Gozes’ discovery of NAP, a snippet of a protein essential for brain formation (activity-dependent neuroprotective protein [ADNP]). As a result of this discovery, a drug candidate that showed efficacy in mild cognitive impairment patients, a precursor to Alzheimer’s disease, is being developed. NAP protects the brain by stabilizing microtubules — tiny cellular cylinders that provide “railways and scaffolding systems” to move biological material within cells and provide a cellular skeleton. Microtubules are of particular importance to nerve cells, which have long processes and would otherwise collapse. In neurodegenerative diseases like Alzheimer’s, the microtubule network falls apart, hindering cellular communication and cognitive function.

"Clinical studies have shown that Davunetide (NAP) protects memory in patients suffering from mild cognitive impairment preceding Alzheimer’s disease," said Prof. Gozes. "While the mechanism was understood in broad terms, the precise molecular target remained a mystery for years. Now, in light of our new research, we know why and we know how to proceed."

Stabilizing microtubules

The breakthrough was the discovery of the mechanism promoting microtubule growth at the tips of the tubes (“rails”). The researchers found that the NAP structure allows it to bind to the tip of the growing microtubule, the emerging “railway,” through specific microtubule end-binding proteins, which adhere to microtubules a bit like locomotors to provide for growth and forward movement, while the other end of the microtubule may to be disintegrating. These growing tips enlist regulatory proteins that are essential for providing plasticity at the nerve cell connection points, the synapses.

"We have now revealed that ADNP through its NAP motif binds the microtubule end binding proteins and enhances nerve cell plasticity, providing for brain resilience. We then discovered that NAP further enhances ADNP microtubule binding," said Prof. Gozes.

Researchers hope their discovery will help move Davunetide (NAP) and related compounds into further clinical trials, increasing the potential of future clinical use. Prof. Gozes is continuing to investigate microtubule end-binding proteins to better understand their protective properties in the brain.