Disease and sleep: Recent studies find new links

One in five U.S. adults shows signs of chronic sleep deprivation, and a shortage of sleep has been linked to health problems as different as diabetes and Alzheimer’s disease. Recent studies have found some interesting connections between illness and what is happening in our brains as we snooze.

Sleep to protect your brain

A new study from Uppsala University, Sweden, shows that one night of sleep deprivation increases morning blood concentrations of NSE and S-100B in healthy young men. These molecules are typically found in the brain. Thus, their rise in blood after sleep loss may indicate that a lack of snoozing might be conducive to a loss of brain tissue. The findings are published in the journal SLEEP.

Fifteen normal-weight men participated in the study. In one condition they were sleep-deprived for one night, while in the other condition they slept for approximately 8 hours.

“We observed that a night of total sleep loss was followed by increased blood concentrations of NSE and S-100B. These brain molecules typically rise in blood under conditions of brain damage. Thus, our results indicate that a lack of sleep may promote neurodegenerative processes”, says sleep researcher Christian Benedict at the Department of Neuroscience, Uppsala University, who lead the study. 

“In conclusion, the findings of our trial indicate that a good night’s sleep may be critical for maintaining brain health”, says Christian Benedict.

Study provides new insights into cause of human neurodegenerative disease

A recent study led by scientists from the National University of Singapore (NUS) opens a possible new route for treatment of Spinal Muscular Atrophy (SMA), a devastating disease that is the most common genetic cause of infant death and also affects young adults. As there is currently no known cure for SMA, the new discovery gives a strong boost to the fight against SMA.

SMA is caused by deficiencies in the Survival Motor Neuron (SMN) gene. This gene controls the activity of various target genes. It has long been speculated that deregulation of some of these targets contributes to SMA, yet their identity remained unknown.

Using global genome analysis, the research team, led by Associate Professor Christoph Winkler of the Department of Biological Sciences at the NUS Faculty of Science and Dr Kelvin See, a former A*STAR graduate scholar in NUS who is currently a Research Fellow at the Genome Institute of Singapore (GIS), found that deficiency in the SMN gene impairs the function of the Neurexin2 gene. This in turn limits the neurotransmitter release required for the normal function of nerve cells. The degeneration of motor neurons in the spinal cord causes SMA. This is the first time that scientists establish an association between Neurexin2 and SMA.

Preliminary experimental data also showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish. This indicates a possible new direction for therapy of neurodegeneration.

Collaborating with Assoc Prof Winkler and the NUS researchers are Dr S. Mathavan and his team at GIS, as well as researchers from the University of Wuerzburg in Germany. The breakthrough discovery was first published in scientific journal Human Molecular Genetics last month.

Small zebrafish provides insights into human neurodegenerative disease

SMA is a genetic disease that attacks a distinct type of nerve cells called motor neurons in the spinal cord. The disease has been found to be caused by a defect in the SMN gene, a widely used gene that is responsible for normal motor functions in the body.

To study how defects in SMN cause neuron degeneration, the scientists utilised a zebrafish model, as the small fish has a relatively simple nervous system that allows detailed imaging of neuron behaviour.

In laboratory experiments, the researchers showed when SMN activity in zebrafish was reduced to levels found in human SMA patients, Neurexin2 function was impaired. This novel disease mechanism was also discovered in other in vivo models, suggesting that it is applicable to mammals and possibly human patients.

When the scientists measured the activity of nerve cells in zebrafish using laser imaging, they found that nerve cells deficient for Neurexin2 or SMN could not be activated to the same level as healthy nerve cells. This impairment consequently led to the reduction of muscular activity. Interestingly, preliminary data showed that a restoration of Neurexin2 activity can partially recover neuron function in SMN deficient zebrafish.

Further studies

Assoc Prof Winkler, who is also with the NUS Centre for Biolmaging Sciences, explained, “These findings significantly advance our understanding of how the loss of SMN leads to neurodegeneration. A better understanding of these mechanisms will lead to novel therapeutic strategies that could aim at restoring and maintaining functions in deficient nerve cells of SMA patients.”

Dr See added, “Our study provides a link between SMN deficiency and its effects on a critical gene important for neuronal function. It would be interesting to perform follow up studies in clinical samples to further investigate the role of Neurexin2 in SMA pathophysiology.”

Moving forward, the team of scientists will conduct further research to determine if Neurexin2 is an exclusive mediator of SMN induced defects and hence can be used as a target for future drug designs. They hope their findings will contribute towards treatment of neurodegeneration.

Alzheimer-substance may be the nanomaterial of tomorrow

It causes brain diseases like Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob’s disease. It is also hard and rigid as steel. Now research at Chalmers University of Technology shows that the amyloid protein carries unique characteristics that may lead to the development of new composite materials for nano processors and data storage of tomorrow and even make objects invisible.

Piotr Hanczyc, PhD student at the department of Chemical and Biological Engineering, shows in an article in Nature Photonics, that the amyloid, a very dense aggregate of protein that causes brain diseases like Alzheimer’s and Parkinson’s, carries unique characteristics. Unlike well-functioning protein the amyloid reacts upon multi photon laser irradiation. This laser may in the future possibly be used for detection of amyloids inside a human brain. This discovery is in itself a breakthrough.

- But you can also create these aggregates in an artificial way in a laboratory and in combination with other materials create unique characteristics, Piotr Hanczyc says.

The amyloid aggregates are as hard and rigid as steel. The difference is that steel is much heavier and has defined material properties whereas amyloids can be tuned for desired purpose. By attaching a material’s molecules to the dense amyloid its characteristics change. This has been known for more than ten years and is already used by scientists.

- What hasn’t been known is that the amyloids react to multi-photon irradiation and this opens up new possibilities to also change the nature of the material attached to the amyloids, Piotr Hanczyc says.

The amyloids are shaped like discs densely piled upon each other.  When a material gets merged with these discs its molecules end up so densely and regularly that they can communicate and exchange information. This means totally new possibilities to change a material’s characteristics.

Multi-photon tests on materials tied to amyloids are yet to be performed, but Piotr sees an opportunity for cooperation with Chalmers material science researchers interested for example in solar cell technology.

And though it may still be science fiction, he also considers that one day scientists may use the material properties of amyloid fibrils in the research of invisible metamaterials.

- An object’s ability to reflect light could be altered so that what’s behind it gets reflected instead of the object itself, in principle changing the index of light refraction, kind of like when light hits the surface of water, Piotr Hanczyc says.

Repairing mitochondria in neurodegenerative disease

The relationship between fine-scale structure and function in the brain is perhaps best explored today by the study of neurodegenerative disease. Disorders like Rett syndrome may be considered developmental in origin—and defined by exotic mechanisms including X-linked inactivation, DNA methylation, and genomic imprinting—but even here, its larger physical pathology evolves through the course of life and continues to be revealed in almost any place that researchers look. When diseases directly involve inputs to the brain like vitamin or diet, and can also be controlled by them, things get even more interesting. More often than not, these disorders have a clear genetic component, are frequently linked to the mitochondria, and lead to progressive and often perplexing deficits of movement. One such enigma is known as pantothenate kinase-associated neurodegeneration, or PKAN syndrome, in its the most frequent form. A recent open paper in the journal Brain explains.

This particular syndrome can be caused by any number of a hundred or so mutations in the PANK2 gene, which codes for the mitochondrial enzyme pantothenate kinase 2. Of the four nuclear-coded PANK genes, only PANK2 is targeted to the mitochondria. Its protein product is involved in co-enzyme A biosynthesis and catalyzes the phosphorylation of pantothenate (vitamin B5). The hallmark pathology, as defined by T2-weighted MRI, can be seen in the globus pallidus and even has its own unique name— the Eye-of-the-Tiger sign.

The researchers used a mouse model of the disease with a Pank2 double gene knockout. On a standard diet, the mice showed growth issues, azoospermia (lack of sperm) and minor mitochondrial dysfunction, but not some of the other typical issues like iron accumulation in the brain or retinal degeneration. Since co-enzyme A is crucial to several metabolic pathways, the researchers also tested the mice on a high fat ketogenic diet. Under these conditions, ketone bodies produced through fatty acid oxidation bypass the normal glycolytic pathways and proceed directly to the citric acid acid.

On the ketogenic diet, the mitochondria, which were already ailing with abnormal, swollen cristae, fared much worse, losing some cristae entirely. Extensive lipofuschin deposits were also found in these mice, and movement issues were amplified. It had previously been established in other organisms like flies, that panthethine (a dimeric form of vitamin B5 linked by cysteamine bridging groups) could counteract these issues. When the mice were given panthethine, the general pathology was resolved. In particular, the mitochondria were completely rescued, presumably restored to health, or otherwise replaced in the natural course of events.

The researchers also evaluated mitochondrial membrane potential using dye staining methods. In the knockout mice, membrane potential was compromised, however it was completely restored by the panthethine. At present there is no definitive way to predict functional variables, like membrane potential, from the morphology as it is seen on processed EM tissue. In a recent review of new brain mapping techniques, we discussed this issue, and also pointed to new technologies which may permit closer examinations.

On EM images, one of the most striking features in the interior of mitochondria is the crista junction. This protein structure functionally divides the inner and intermembrane spaces, and controls exchanges between them. While mitochondria come in a variety of forms, the junctions generally converge on a preferred shape and size. Efforts to thermodynamically characterize them in terms of shape entropy have been initiated, as have conceptions of how they evolve as conditions in the mitochondria change mechanically. The so-called “baffle model” of mitochondrial has been entirely replaced by the new cristae junction model which aims to relate structure to function for these organelles, just as we seek it on larger scales for the brain.

Several issues in PNAK style neurodegeneration still stand out like a sore thumb. The iron accumulation is still unexplained, but may be related to another unexplained issue: namely, not only does panthethine fail to cross the BBB, it does not even appear to be working through a vitamin B5 function. When panthethine is metabolized into two pantothenic acid molecules, it also forms two cysteamines. While cysteamine is associated with various side effects, and it can bind and inactivate certain liver enzymes, it also can cross the BBB, perhaps as seen here, to great effect.

The doses necessary for vitamin B5 function are far below those needed here for restorative function. More work is needed to constrain the range of possible mechanisms at play here, but in addition to finding cures for the disease, it will also help cure our ignorance as far as structure-function relations.

How Our Nerves Keep Firing

University of Utah and German biologists discovered how nerve cells recycle tiny bubbles or “vesicles” that send chemical nerve signals from one cell to the next. The process is much faster and different than two previously proposed mechanisms for recycling the bubbles.

Researchers photographed mouse brain cells using an electron microscope after flash-freezing the cells in the act of firing nerve signals. That showed the tiny vesicles are recycled to form new bubbles only one-tenth of a second after they dump their cargo of neurotransmitters into the gap or “synapse” between two nerve cells or neurons.

“Without recycling these containers or ‘synaptic vesicles’ filled with neurotransmitters, you could move once and stop, think one thought and stop, take one step and stop, and speak one word and stop,” says University of Utah biologist Erik Jorgensen, senior author of the study in the Dec. 4 issue of the journal Nature.

“A fast nervous system allows you to think and move. Recycling synaptic vesicles allows your brain and muscles to keep working longer than a couple of seconds,” says Jorgensen, a distinguished professor of biology. “This process also may protect neurons from neurodegenerative diseases like Lou Gehrig’s disease and Alzheimer’s. So understanding the process may give us insights into treatments someday.”

A brain cell maintains a supply of 300 to 400 vesicles to send chemical nerve signals, using up to several hundred per second to release neurotransmitters, says the study’s first author, postdoctoral fellow Shigeki Watanabe.

Recycling vesicles is called “endocytosis.” Jorgensen and Watanabe named the process they observed “ultrafast endocytosis.” They showed it takes one-tenth of a second for a vesicle to be recycled, and such recycling occurs on the edge of “active zone” – the place on the end of the nerve cell where the vesicles first unload neurotransmitters into the synapse between brain cells.

“It’s like Whac-A-Mole: one vesicle goes down (fuses and unloads) and another pops up someplace else,” Jorgensen says.

Jorgensen believes ultrafast endocytosis is the most common way of recycling vesicles, but says the study doesn’t disprove two other, long-debated hypotheses:

– “Kiss-and-run endocytosis,” which supposedly takes one second, with a vesicle just “kissing” the inside of its nerve cell, dumping its neurotransmitters outside and “running” by detaching to reform a recycled vesicle in the same part of the active zone.

– Clathrin-mediated endocytosis,” which purportedly takes 20 seconds and occurs away from the active zone, at a point where a protein named clathrin assembles itself into a soccer-ball-shaped scaffold that forms a new vesicle or bubble.

Earlier this year, Jorgensen, Watanabe and colleagues published a related study in the journal eLife revealing that ultrafast endocytosis occurs in nematode worms. The new study of hippocampal brain cells from mice “tells us that mammals – and thus humans – do it the same way,” Jorgensen says. “The two papers together identify a process never previously seen – much faster than has been measured before.”

Jorgensen and Watanabe conducted the study with M. Wayne Davis, a University of Utah research assistant professor of biology; and technician Berit Söhl-Kielczynski and neuroscientists Christian Rosenmund, Benjamin Rost and Marcial Camacho-Pérez, all of Germany’s Charity University Medicine Berlin.

The study was funded by the National Institutes of Health, the European Research Council and the German Research Council. Jorgensen also is funded by his status as a Howard Hughes Medical Institute investigator and an Alexander von Humboldt Scholar.

Machine Gun Analogy for Vesicle Recycling

The process of a vesicle fusing to the nerve cell’s wall from the inside, then releasing neurotransmitters into the synapse is known as “exocytosis.” An analogy might be a bubble rising from boiling soup and releasing steam. The liquid part of the bubble fuses with the liquid in the soup, sooner or later to arise in another bubble.

The 2013 Nobel Prize in Physiology or Medicine went to three scientists who discovered key aspects of vesicle transport of cargo and exocytosis in nerve and other cells: which genes are required for vesicle transport, how vesicles deliver cargo to the correct locations, and how vesicles in brain cells release neurotransmitters to send a signal to the next brain neuron.

Jorgensen, Shigeki and colleagues studied the next step, endocytosis: how the membrane that forms vesicles (and nerve cell walls) is recycled to form new vesicles.

To illustrate the three possible mechanisms for recycling vesicles, Jorgensen compares vesicles with machine gun shells.

“You are fusing vesicles to the nerve cell membrane and expelling the neurotransmitter contents at extremely high rates,” he says. “The synapse will use up its ‘ammo’ very quickly at these rates, so the cell needs to refill the empty shells.”

Clathrin-mediated vesicle recycling is like “remaking the shell from scratch,” he says, while kiss-and-run endocytosis is like picking up every empty shell casing and refilling them one at a time.

“Ultrafast endocytosis allows the synapse to whip up all of the empty shells by the handful, fill them, and put them back in line at incredibly fast rates so the machine gun never runs out of ammo,” Jorgensen says.

Flash and Freeze for Nerve Cells in Action

Shigeki, Jorgensen and colleagues developed a method to photograph the tiny vesicles inside a nerve cell as the bubbles moved to the end of the cell, fused with the cell membrane, dumped their load of neurotransmitters into the gap or “synapse” between nerve cells, and then were recycled to reappear as new bubbles inside the nerve cell.

“We found a way to look at this process on a timescale that no one ever looked at before,” Watanabe says.

First, the researchers grew hundreds of brain cells from the mouse hippocampus – the often-studied part of the brain required for memory formation – on quarter-inch-wide sapphire disks placed in petri dishes with growth medium.

They added an algae gene to mouse brain cells that made the neurons produce an “ion channel” – basically a switch – that is stimulated by light instead of electricity. Then the brain cells were placed in a super-cold, high-pressure chamber, at 310 degrees below zero Fahrenheit and pressure 2,000 times greater than Earth’s atmosphere at sea level.

A wire cannot be routed into the chamber, which is why the cells were genetically programmed to be stimulated by light. The researchers flashed blue light on the mouse brain cells, making them “fire” neurotransmitter nerve signals. At the same time, the firing neurons were frozen with a blast of liquid nitrogen. To catch neurons in all stages of firing, the nerve cells were frozen at various times after the flash of blue light: 15, 30 and 100 milliseconds and one, three and 10 seconds.

“We built a new device to capture neurons performing fast behaviors,” Jorgensen says. “It stops all motion in the cell – even membranes in the act of fusing.

“We call it flash and freeze,” Watanabe says.

Next, the sapphire disks with neurons were put into liquid epoxy, which hardened and then were thin-sliced so the neurons could be photographed under an electron microscope. The ultrafast formation of recycled vesicles was visible.

“You see the outline of the membrane,” Jorgensen says. “You see the bubbles or vesicles in different stages of formation.”

Watanabe says about 3,000 mouse brain cell synapses were flashed, frozen and analyzed during the study. About 20 percent of the nerve cells had been fired and showed signs that nerve vesicles were being recycled.