Researchers discover how brain cells change their tune

Brain cells talk to each other in a variety of tones. Sometimes they speak loudly but other times struggle to be heard. For many years scientists have asked why and how brain cells change tones so frequently. Today National Institutes of Health researchers showed that brief bursts of chemical energy coming from rapidly moving power plants, called mitochondria, may tune brain cell communication.

"We are very excited about the findings," said Zu-Hang Sheng, Ph.D., a senior principal investigator and the chief of the Synaptic Functions Section at the NIH’s National Institute of Neurological Disorders and Stroke (NINDS). "We may have answered a long-standing, fundamental question about how brain cells communicate with each other in a variety of voice tones."

The network of nerve cells throughout the body typically controls thoughts, movements and senses by sending thousands of neurotransmitters, or brain chemicals, at communication points made between the cells called synapses. Neurotransmitters are sent from tiny protrusions found on nerve cells, called presynaptic boutons. Boutons are aligned, like beads on a string, on long, thin structures called axons. They help control the strength of the signals sent by regulating the amount and manner that nerve cells release transmitters.

Mitochondria are known as the cell’s power plant because they use oxygen to convert many of the chemicals cells use as food into adenosine triphosphate (ATP), the main energy that powers cells. This energy is essential for nerve cell survival and communication. Previous studies showed that mitochondria can rapidly move along axons, dancing from one bouton to another.

In this study, published in Cell Reports, Dr. Sheng and his colleagues show that these moving power plants may control the strength of the signals sent from boutons.

"This is the first demonstration that links the movement of mitochondria along axons to a wide variety of nerve cell signals sent during synaptic transmission," said Dr. Sheng.

The researchers used advanced microscopic techniques to watch mitochondria move among boutons while they released neurotransmitters. They found that boutons sent consistent signals when mitochondria were nearby.

"It’s as if the presence of mitochondria causes a bouton to talk in a monotone voice," said Tao Sun, Ph.D., a researcher in Dr. Sheng’s laboratory and the first author of the study.

Surprisingly, when the mitochondria were missing or moving away from boutons, the signal strength fluctuated. The results suggested that the presence of stationary power plants at synapses controls the stability of the nerve signal strength.

To test this idea further, the researchers manipulated mitochondrial movement in axons by changing levels of syntaphilin, a protein that helps anchor mitochondria to the nerve cell’s skeleton found inside axons. Removal of syntaphilin resulted in faster moving mitochondria and electrical recordings from these neurons showed that the signals they sent fluctuated greatly. Conversely, elevating syntaphilin levels in nerve cells arrested mitochondrial movement and resulted in boutons that spoke in monotones by sending signals with the same strength.

"It’s known that about one third of all mitochondria in axons move. Our results show that brain cell communication is tightly controlled by highly dynamic events occurring at numerous tiny cell-to-cell connection points," said Dr. Sheng.

In separate experiments the researchers watched ATP energy levels in these tiny boutons as they sent nerve messages.

"The levels fluctuated more in boutons that did not have mitochondria nearby," said Dr. Sun.

The researchers also found that blocking ATP production in mitochondria with the drug oligomycin reduced the size of the signals boutons sent even if a mitochondrial power plant was nearby.

"Our results suggest that local ATP production by nearby mitochondria is critical for consistent neurotransmitter release," said Dr. Sheng. "It appears that variability in synaptic transmission is controlled by rapidly moving mitochondria which provide brief bursts of energy to the boutons they pass through."

Problems with mitochondrial energy production and movement throughout nerve cells have been implicated in Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and other major neurodegenerative disorders. Dr. Sheng thinks these results will ultimately help scientists understand how these problems can lead to disorders in brain cell communication.

"Our findings reveal the cellular mechanisms that tune brain communication by regulating mitochondrial mobility, thus advancing our understanding of human neurological disorders," said Dr. Sheng.

Peering into the Protein Pathways of a Cell

As a cell’s central power plant, the mitochondrion is a busy place.

Specially-coded proteins from the nucleus are constantly being ferried across the mitochondrion’s inner membrane, where they help the mighty organelle do its work – producing the cell’s high-energy molecules, carrying out signaling duties, and controlling cell growth.

Scientists have long known that the central channel through which most of these proteins must pass – a critical gatekeeper known as the translocase of the inner mitochondrial membrane 23 or TIM23 for short – requires an electrical field for its gating capabilities to function. But they weren’t quite sure how the whole process worked.

Until now.

Using highly sensitive fluorescent probes, a team of scientists based at UConn has managed to peer deep into the inner workings of a cell, capturing the never-before-seen structural dynamics of the TIM23 channel complex while it functioned in its natural environment.

In doing so, the team, led by Nathan N. Alder, an assistant professor in the Department of Molecular and Cell Biology in the College of Liberal Arts and Sciences, discovered that the TIM23 complex not only opens and closes in response to fluctuations in the energized state of the mitochondrion’s inner membrane, as the scientific community suspected, it also changes its very structure – altering the helical shape of protein segments that line the channel – as the electrical field across the membrane drops.

The research, which appears this week in the peer-reviewed journal Nature Structural & Molecular Biology, explains how the energized state of the membrane drives the structural dynamics of membrane proteins and sheds new light on how cellular transport systems harness energy to perform their work inside the cell. It also shows how fluorescent mapping at the subcellular level may reveal new insights into the underlying causes of neurodegenerative and metabolic disorders associated with mitochondrial function.

In an overview of the research accompanying the paper’s publication, Nikolaus Pfanner of the University of Freiburg, Germany, an international leader in the field of cellular protein trafficking, and several members of his research group, called the study “a major step towards a molecular understanding of a voltage-gated protein translocase.”

“The molecular nature of voltage sensors in membrane proteins is a central question in biochemical research,” Pfanner and his colleagues said. “The study … is not only of fundamental importance for our understanding of mitochondrial biogenesis, but also opens up new perspectives in the search for voltage-responsive elements in membrane proteins.”

Applying a new technique

The fluorescent mapping technique used in the research was a key to the project’s success. Alder says he first realized the application’s potential when he successfully mapped channel proteins in a functioning mitochondrion in 2008. In the current study, he advanced the process further, using probes to capture the behavior of a particular segment of the TIM23 channel complex as it was impacted by voltage changes in the membrane’s electrical field.

“Fluorescent mapping made this possible,” says Alder, who, as a post-doctoral student, worked with protein fluorescent labeling pioneer Arthur E. Johnson of Texas A&M’s Health Science Center. “It allowed us to peer into the functioning dynamics of a protein import channel complex that is responsible for building up the power plant of the cell … What we found was that these protein-trafficking complexes are certainly not static. This is a very, very dynamic channel.”

To monitor the fluorescence probes inside the mitochondria, the research team used advanced spectrofluorimeters equipped with xenon lamps and laser diodes to measure steady-state and time-resolved fluorescence, respectively.

To conduct the study, Alder incorporated cysteine residues modified with a fluorescent probe at specific positions along a transmembrane segment of a TIM23 complex derived from a common species of yeast, Saccharomyces cerevisiae. The team then monitored the probes in real time to observe how the channel’s voltage-gating and structure responded to induced changes in the inner membrane’s electrical field.

“It’s an indirect way of looking at the structure of something, but because we are able to look into an actually functioning mitochondrion, it’s given us a whole world of new information,” says Alder.

“That the magnitude of the voltage gradient across the membrane could play a significant role in defining the structure of these proteins is probably one of the most significant elements of this research,” he adds.

A defining moment

Watching the process was, for Alder, a defining moment in his professional career.

“When I first saw a certain kind of structure that told me I was in the middle of a channel, that was one of the most exciting times in my professional life,” he says. “I knew I was getting insight into a fundamental natural phenomenon, something no one has ever seen before.”

When Alder saw the protein-conducting channel bending and collapsing in response to changes in the membrane’s voltage levels, he was equally thrilled.

“That was one of those rare technical moments in my professional life that showed we were really getting insight into a fundamental process going on inside a cell,” he says. “It’s always been known that you need an energized membrane to make these channels work, but no one had a clue why.”

Joining Alder on the project were UConn graduate students Ketan Malhotra and Murugappan Sathappa and research associate Judith S. Landin. Johnson, Alder’s former mentor at Texas A&M, is also listed as a co-author. The work in the Alder Lab was funded by the National Science Foundation; work done in the Johnson Lab was additionally sponsored by the National Institutes of Health and the Robert A. Welch Foundation.

Alder says the next phase of the research will look toward isolating the TIM23 protein channel complex in an artificial system to see if it continues to respond to voltage fluctuations outside of its natural habitat. The research team is also hoping to identify the particular parts of the protein complex that are acting as voltage sensors.

“Once we start to identify exactly what is the voltage sensor, we will have a better understanding of the translocase process, and ultimately we can apply this knowledge to other kinds of protein transporters whose dysfunction has been implicated in the etiology of diseases such as cardiovascular disease and cancer,” Alder says. “If their function is tied to the energized state of the membrane, we’ll be able to see whether defects in that ability to couple to the membrane might be associated with the pathogenesis of these diseases.”

Balancing mitochondrial dynamics in Alzheimer’s disease

Many diseases are multifactorial and can not be understood by simple molecular associations alone. Alzheimer’s disease (AD) is associated with toxic transformations in two classes of protein,amyloid beta and tau, but they do not explain the full underlying pathology. On the cellular scale, much of the real-time morphological changes in neurons can be attributed to their underlying mitochondrial dynamics—namely fission, fusion, and the motions between these events. Last year, researchers from Harvard Medical School made the intriguing discovery that alterations in tau could lead to a doubling in the length of mitochondria. This week, they published a review article in Trends in Neuroscience, in which they seek to explain the primary features of AD in terms of mitochondrial dynamics.

Together with a collaborator from the Queensland Brain Institute, the Harvard researchers arrive at the conclusion that, like many other neurological diseases, AD is fundamentally an energy problem. While some proteins, like APOE-ɛ4 can predispose one to AD, point defects in individual proteins can not account for AD in the same way that a single alteration in hemoglobin leads to sickle cell disease. Attempts to assign casual relations to the complex interactions of tau or amyloid, with hundreds of other proteins inside neurons have frequently served to cloud, rather than simplify the AD story.

In years gone by, it was possible to publish a paper about how phosphorylation at certain sites on proteins, like tau, could lead to any number of downstream events. Tau is one of many proteins that control the assembly and stability of microtubules, critical structures that are among those compromised in AD. The problem now, is that we know tau comes in so many flavors—it is a big family of different isoforms with different properties depending on how they are processed. As far as simple phosphorylation, tau has been found to have 79 potential sites, with at least 30 of them normally phosphorylated.

A welcome simplification to this situation of compounding molecular complexity, is that many pathways converge onto convenient pre-existing packets of time, space, and predictable molecular structure—the mitochondria. As opposed to massive cell-wide molecular accounting, describing a few sub-cellular morphological features may be a more tractable approach not only to capture disease etiology, but perhaps to treat it.

To this end, the researchers apply existing knowledge regarding some of the molecular players in AD, to a few of the well-established control points in mitochondrial dynamics. State transitions between fission and fusion are, at the moment at least, characterized by only a small handful of proteins. This simple formula might be prescribed as the following: molecular pathway locally effects the organelle dynamics, then, the dynamic behavior of organelle accounts for the disease. The imposition of this middleman can potentially simplify much of the vast body of fact and conjecture associated with the disease.

The elongation of mitochondria by tau can be caused by increasing fusion, decreasing fission, or both. One function of tau is to stabilize F-actin networks which prevents a key fission protein from ever reaching the mitochondria. Elongated mitochondria do not necessarily cause AD. In fact, amyloid beta, which is concentrated inside mitochondria, has been shown to cause increased fission and decreased fusion. When the balance between fission and fusion is pushed too far in either direction, the result is bad news for neurons. If there are defects in the transport of mitochondria, as seems to be the case in many neurological diseases, their redistribution is unable to compensate for this loss of balance.

Specific disease-associated isoforms and phosphorylation states of tau can lead to AD through the loss of mitochondria in axons. In studies of AD tissue, mitochondria have been found to be preferentially redistributed to the soma. These selective localizations can take place quickly, and are therefore difficult to quantify except by live videomicroscopy. In synapses, the mitochondria have been observed to be longer lived, and to play a more critical role in calcium regulation then those elsewhere. Disruption in the normal handling of calcium has been attributed to many aspects of AD, particularly synaptic pathology.

The canonical dogma that action potentials lead to vesicle fusion and transmitter release exclusively through the entry of extracellular calcium has recently been enhanced with the understanding that mitochondria contribute significantly to the synaptic calcium cycle. While mitochondria clearly do not depolarize as rapidly as whole spiking cells,(generally when mitochondria are depolarized there is some problem) their calcium transporters operate quickly to mop up and redistribute calcium. To say that mitochondria might single-handedly initiate vesicle fusion, or for that matter minipotentials or full-blown spikes, would await future experimental corroboration.

Countless scores of papers over the years have attempted to make sense of the myriad synaptic pathways underlying memory and LTP. They might be better understood when mitochondria are viewed as the primary authors of synaptic vesicle release probability, and by implication, “spontaneous” release (vesicle fusion in the absence of a spike). As in disease states, specific pathways, structures and organelles have significant roles to play in many aspects of brain function—but causally relating the motions and dynamics of mitochondria to these phenomena now gives the broadest interpretive power.

Missing link in Parkinson’s disease found

Researchers at Washington University School of Medicine in St. Louis have described a missing link in understanding how damage to the body’s cellular power plants leads to Parkinson’s disease and, perhaps surprisingly, to some forms of heart failure.

These cellular power plants are called mitochondria. They manufacture the energy the cell requires to perform its many duties. And while heart and brain tissue may seem entirely different in form and function, one vital characteristic they share is a massive need for fuel.

Working in mouse and fruit fly hearts, the researchers found that a protein known as mitofusin 2 (Mfn2) is the long-sought missing link in the chain of events that control mitochondrial quality.

The findings are reported April 26 in the journal Science.

The new discovery in heart cells provides some explanation for the long known epidemiologic link between Parkinson’s disease and heart failure.

“If you have Parkinson’s disease, you have a more than two-fold increased risk of developing heart failure and a 50 percent higher risk of dying from heart failure,” says senior author Gerald W. Dorn II, MD, the Philip and Sima K. Needleman Professor of Medicine. “This suggested they are somehow related, and now we have identified a fundamental mechanism that links the two.”

Heart muscle cells and neurons in the brain have huge numbers of mitochondria that must be tightly monitored. If bad mitochondria are allowed to build up, not only do they stop making fuel, they begin consuming it and produce molecules that damage the cell. This damage eventually can lead to Parkinson’s or heart failure, depending on the organ affected. Most of the time, quality-control systems in a healthy cell make sure damaged or dysfunctional mitochondria are identified and removed.

Over the past 15 years, scientists have described much of this quality-control system. Both the beginning and end of the chain of events are well understood. And since 2006, scientists have been working to identify the mysterious middle section of the chain – the part that allows the internal environment of sick mitochondria to communicate to the rest of the cell that it needs to be destroyed.

“This was a big question,” Dorn says. “Scientists would draw the middle part of the chain as a black box. How do these self-destruct signals inside the mitochondria communicate with proteins far away in the surrounding cell that orchestrate the actual destruction?”

“To my knowledge, no one has connected an Mfn2 mutation to Parkinson’s disease,” Dorn says. “And until recently, I don’t think anybody would have looked. This isn’t what Mfn2 is supposed to do.”

Mitofusin 2 is known for its role in fusing mitochondria together, so they might exchange mitochondrial DNA in a primitive form of sexual reproduction.

“Mitofusins look like little Velcro loops,” Dorn says. “They help fuse together the outer membranes of mitochondria. Mitofusins 1 and 2 do pretty much the same thing in terms of mitochondrial fusion. What we have done is describe an entirely new function for Mfn2.”

The mitochondrial quality-control system begins with what Dorn calls a “dead man’s switch.”

“If the mitochondria are alive, they have to do work to keep the switch depressed to prevent their own self-destruction,” Dorn says.

Specifically, mitochondria work to import a molecule called PINK. Then they work to destroy it. When mitochondria get sick, they can’t destroy PINK and its levels begin to rise. Then comes the missing link that Dorn and his colleague Yun Chen, PhD, senior scientist, identified. Once PINK levels get high enough, they make a chemical change to Mfn2, which sits on the surface of mitochondria. This chemical change is called phosphorylation. Phosphorylated Mfn2 on the surface of the mitochondria can then bind with a molecule called Parkin that floats around in the surrounding cell.

Once Parkin binds to Mfn2 on sick mitochondria, Parkin labels the mitochondria for destruction. The labels then attract special compartments in the cell that “eat” and destroy the sick mitochondria. As long as all links in the quality-control system work properly, the cells’ damaged power plants are removed, clearing the way for healthy ones.

“But if you have a mutation in PINK, you get Parkinson’s disease,” Dorn says. “And if you have a mutation in Parkin, you get Parkinson’s disease. About 10 percent of Parkinson’s disease is attributed to these or other mutations that have been identified.”

According to Dorn, the discovery of Mfn2’s relationship to PINK and Parkin opens the doors to a new genetic form of Parkinson’s disease. And it may help improve diagnosis for both Parkinson’s disease and heart failure.

“I think researchers will look closely at inherited Parkinson’s cases that are not explained by known mutations,” Dorn says. “They will look for loss of function mutations in Mfn2, and I think they are likely to find some.”

Similarly, as a cardiologist, Dorn and his colleagues already have detected mutations in Mfn2 that appear to explain certain familial forms of heart failure, the gradual deterioration of heart muscle that impairs blood flow to the body. He speculates that looking for mutations in PINK and Parkin might be worthwhile in heart failure as well.

“In this case, the heart has informed us about Parkinson’s disease, but we may have also described a Parkinson’s disease analogy in the heart,” he says. “This entire process of mitochondrial quality control is a relatively small field for heart specialists, but interest is growing.”

Couch Potatoes May Be Genetically Predisposed to Being Lazy

Studies show 97 percent of American adults get less than 30 minutes of exercise a day, which is the minimum recommended amount based on federal guidelines. New research from the University of Missouri suggests certain genetic traits may predispose people to being more or less motivated to exercise and remain active. Frank Booth, a professor in the MU College of Veterinary Medicine, along with his post-doctoral fellow Michael Roberts, were able to selectively breed rats that exhibited traits of either extreme activity or extreme laziness. They say these rats indicate that genetics could play a role in exercise motivation, even in humans.

“We have shown that it is possible to be genetically predisposed to being lazy,” Booth said. “This could be an important step in identifying additional causes for obesity in humans, especially considering dramatic increases in childhood obesity in the United States. It would be very useful to know if a person is genetically predisposed to having a lack of motivation to exercise, because that could potentially make them more likely to grow obese.”

In their study published in the American Journal of Physiology: Regulatory, Integrative and Comparative Physiology on April 3, 2013, Roberts and Booth put rats in cages with running wheels and measured how much each rat willingly ran on their wheels during a six-day period. They then bred the top 26 runners with each other and bred the 26 rats that ran the least with each other. They repeated this process through 10 generations and found that the line of running rats chose to run 10 times more than the line of “lazy” rats.

Once the researchers created their “super runner” and “couch potato” rats, they studied the levels of mitochondria in muscle cells, compared body composition and conducted thorough genetic evaluations through RNA deep sequencing of each rat.

“While we found minor differences in the body composition and levels of mitochondria in muscle cells of the rats, the most important thing we identified were the genetic differences between the two lines of rats,” Roberts said. “Out of more than 17,000 different genes in one part of the brain, we identified 36 genes that may play a role in predisposition to physical activity motivation.”

Now that the researchers have identified these specific genes, they plan on continuing their research to explore the effects each gene has on motivation to exercise.