In a new study, scientists at the National Institutes of Health took a molecular-level journey into microtubules, the hollow cylinders inside brain cells that act as skeletons and internal highways. They watched how a protein called tubulin acetyltransferase (TAT) labels the inside of microtubules. The results answer long-standing questions about how TAT tagging works and offer clues as to why it is important for brain health.
Microtubules are constantly tagged by proteins in the cell to designate them for specialized functions, in the same way that roads are labelled for fast or slow traffic or for maintenance. TAT coats specific locations inside the microtubules with a chemical called an acetyl group. How the various labels are added to the cellular microtubule network remains a mystery. Recent findings suggested that problems with tagging microtubules may lead to some forms of cancer and nervous system disorders, including Alzheimer’s disease, and have been linked to a rare blinding disorder and Joubert Syndrome, an uncommon brain development disorder.
‘This is the first time anyone has been able to peer inside microtubules and catch TAT in action,’ said Antonina Roll-Mecak, Ph.D., an investigator at the NIH’s National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, Maryland, and the leader of the study.
Microtubules are found in all of the body’s cells. They are assembled like building blocks, using a protein called tubulin. Microtubules are constructed first by aligning tubulin building blocks into long strings. Then the strings align themselves side by side to form a sheet. Eventually the sheet grows wide enough that it closes up into a cylinder. TAT then bonds an acetyl group to alpha tubulin, a subunit of the tubulin protein.
Some microtubules are short-lived and can rapidly change lengths by adding or removing tubulin pieces along one end, whereas others remain unchanged for longer times. Recognising the difference may help cells function properly. For example, cells may send cargo along stable microtubules and avoid ones that are being rebuilt. Cells appear to use a variety of chemical labels to describe the stability of microtubules.
‘Our study uncovers how TAT may help cells distinguish between stable microtubules and ones that are under construction,’ said Dr. Roll-Mecak. According to Dr. Roll-Mecak, high levels of microtubule tagging are unique to nerve cells and may be the reason that they have complex shapes allowing them to make elaborate connections in the brain.
For decades scientists knew that the insides of long-lived microtubules were often tagged with acetyl groups by TAT. Changes in acetylation may influence the health of nerve cells. Some studies have shown that blocking this form of microtubule tagging leads to nerve defects, brain abnormalities or degeneration of nerve fibres. Since the discovery of microtubule acetylation, scientists have been puzzled about how TAT accesses the inside of the microtubules and how the tagging reaction happens.
To watch TAT at work, Dr. Roll-Mecak and her colleagues took high resolution movies of individual TAT molecules interacting with microtubules in real time. They saw that TAT surfs through the inside of microtubules and although it can find acetylation sites quickly, the process of adding the tag occurs very slowly.
In general, tagging reactions work like keys fitting into locks: the better the key fits, the faster the lock can open. Similarly, the rate of the reactions is determined by how well TAT molecules fit around tagging sites.
Dr. Roll-Mecak’s team investigated this idea by using a technique called X-ray crystallography to look at how atoms on TAT molecules interact with acetylation sites on tubulin molecules. Their results suggested that TAT fit poorly around the sites.
‘It looks as though TAT can easily journey through microtubules spotting acetylation sites but may only label those that are stable for longer periods of time,’ said Dr. Roll-Mecak.
This may help cells identify the microtubules they need to rapidly change shapes or send cargo to other places. Further studies may help researchers understand how microtubule tagging influences nerve cells in health and disease. National Institute of Neurological Disorders and Stroke

This spring has brought rare but tangible moments of progress against the devastating lung disease idiopathic pulmonary fibrosis (IPF), which afflicts millions of people worldwide. Two drugs recently showed promise in clinical trials, and now a study offers both an unprecedentedly deep explanation of how the disease progresses and introduces another potential therapeutic avenue.
The new study features a central figure: an evolutionarily ancient protein called ‘chitinase 3-like-1’ (CHI3L1). The authors implicate it as the ‘master regulator’ of what appears to be a tragically errant repair response to the mysterious lung injuries that give rise to the disease. In describing how CHI3L1 works in IPF, the research also points to a strategy for treatment.
The report demonstrates that CHI3L1 is produced to help in response to the injury. It feeds back to protect injured cells from dying and simultaneously stimulates tissue repair to patch the damage that has occurred. But the study also shows how this dual role contributes to the ultimate problem. If IPF resulted from a single injury, like a paper cut, CHI3L1 would decrease the injury and cause local scarring while it restored tissue integrity. In that case, the amount of scarring would not be excessive and tissue function would not be significantly altered. But in IPF lungs, cells undergo ongoing injury, so CHI3L1 is chronically elevated and scar tissue accumulates. As CHI3L1 rescues cell after cell, the scarring builds up, eventually compromising the lung’s ability to breathe. In IPF, 70 percent of patients die within five years.
‘The CHI3L1 is doing exactly what it is supposed to do — it is designed to shut off cell death and decrease injury,’ said Dr. Jack A. Elias, a co-senior author of the study and dean of medicine and biological sciences at Brown University. He is joined on the paper by a host of his former colleagues and students at Yale University where the research occurred. ‘But at the same time it is decreasing cell death it is driving the fibrosis. You’ve got this ongoing injury so you’ve got these ongoing attempts to shut off injury which stimulate scarring.’
　
　
They compared tissues and serum from normal patients, outpatients with IPF, and patients with an acute exacerbation (AE) of IPF. In AE, widespread lung injury is superimposed on the pulmonary fibrosis, which frequently occurs before patients die. In lung biopsies and serum, they found that CHI3L1 levels are elevated in both tissue compartments in the outpatients with IPF and that the levels of CHI3L1 correlated with their disease progression. In the patients with AE, elevated levels of CHI3L1 were not noted, showing that the levels of CHI3L1 decrease right before the patients die.
‘This demonstrates that the CHI3L1 plays a key role in controlling lung injury in this setting,’ Elias said.
After documenting that elevated levels of CHI3L1 correlate with ongoing fibrosis and scarring and that a lack of the protein associates with widespread cell death, the team engaged in several manipulations of CHI3L1 in mice to see how levels and the clinical outcomes might be related. (In mice, CHI3L1 is also called BRP-39.)
Scientists can induce an IPF-like response in mice using a drug called bleomycin. In mice given bleomycin, the researchers found that the levels of CHI3L1 declined at first and then surged. At the times when the protein levels were low, cell damage occurred, and when the protein surged, the excessive scarring set in.
In previous research the team had engineered several lines of genetically modified mice. Some were transgenic and can produce CHI3l1 on chemically delivered command. Other mice were engineered to never produce BRP-39 — the mouse version of CHI3L1 — at all.
Using these mice, the researchers found that if they triggered CHI3L1 production early after administering bleomycin, the mice fared well, experiencing less injury, less damage and less scarring than controls. If they waited several days after bleomycin to trigger CHI3L1, the mice fared very poorly and scarring and mortality went up.
Mice who couldn’t produce CHI3L1/BRP-39, had acute lung cell damage, somewhat like AE patients who have a relative deficiency of CHI3L1. However, without CHI3L1 they did not generate much scarring.
All of these findings were supplemented with several other experiments that were designed to learn how CHI3L1 interacts with other cells involved in the tissue repair response in both human and mouse lungs. The experiments, including studies conducted in a bioengineered 3-D model of lung tissue seeded with relevant cells, showed that CHI3L1 regulates a pathway that recruits cells such as macrophages and fibroblasts that produce the scarring, or fibrosis.
In all, the results show that CHI3L1 plays a fundamental role in the course, if not the origin, of IPF. An ongoing buildup of it results in excessive scarring. Too little and cells die much more frequently.
‘To my knowledge this is the first comprehensive paper that’s been able to explain the many facets and presentations of IPF,’ Elias said. ‘It explains and links the injury and the repair responses that are critical in the disease. It also provides an explanation for the slowly progressing patients and the patients that experience acute exacerbations.’ Brown University

A new drug target to fight Alzheimer’s disease has been discovered by a research team led by Gong Chen, a professor of biology and the Verne M. Willaman Chair in Life Sciences at Penn State. The discovery also has potential for development as a novel diagnostic tool for Alzheimer’s disease, which is the most common form of dementia and one for which no cure has yet been found.

Chen’s research was motivated by the recent failure in clinical trials of once-promising Alzheimer’s drugs being developed by large pharmaceutical companies. ‘Billions of dollars were invested in years of research leading up to the clinical trials of those Alzheimer’s drugs, but they failed the test after they unexpectedly worsened the patients’ symptoms,’ Chen said.

The research behind those drugs had targeted the long-recognised feature of Alzheimer’s patients’ brains: the sticky buildup of the amyloid protein known as plaques, which can cause neurons in the brain to die.

‘The research of our lab and others now has focused on finding new drug targets and on developing new approaches for diagnosing and treating Alzheimer’s disease,’ Chen explained.

‘We recently discovered an abnormally high concentration of one inhibitory neurotransmitter in the brains of deceased Alzheimer’s patients,’ Chen said.

He and his research team found the neurotransmitter, called GABA (gamma-aminobutyric acid), in deformed cells called ‘reactive astrocytes’ in a structure in the core of the brain called the dentate gyrus. This structure is the gateway to hippocampus, an area of the brain that is critical for learning and memory.

Chen’s team found that the GABA neurotransmitter was drastically increased in the deformed versions of the normally large, star-shaped ‘astrocyte’ cells which, in a healthy individual, surround and support individual neurons in the brain. ‘Our research shows that the excessively high concentration of the GABA neurotransmitter in these reactive astrocytes is a novel biomarker that we hope can be targeted in further research as a tool for the diagnosis and treatment of Alzheimer’s disease,’ Chen said.

Chen’s team developed new analysis methods to evaluate neurotransmitter concentrations in the brains of normal and genetically modified mouse models for Alzheimer’s disease (AD mice).

‘Our studies of AD mice showed that the high concentration of the GABA neurotransmitter in the reactive astrocytes of the dentate gyrus correlates with the animals’ poor performance on tests of learning and memory,’ Chen said.

His lab also found that the high concentration of the GABA neurotransmitter in the reactive astrocytes is released through an astrocyte-specific GABA transporter, a novel drug target found in this study, to enhance GABA inhibition in the dentate gyrus. With too much inhibitory GABA neurotransmitter, the neurons in the dentate gyrus are not fired up like they normally would be when a healthy person is learning something new or remembering something already learned.

Importantly, Chen said, ‘After we inhibited the astrocytic GABA transporter to reduce GABA inhibition in the brains of the AD mice, we found that they showed better memory capability than the control AD mice. We are very excited and encouraged by this result because it might explain why previous clinical trials failed by targeting amyloid plaques alone. One possible explanation is that while amyloid plaques may be reduced by targeting amyloid proteins, the other downstream alterations triggered by amyloid deposits, such as the excessive GABA inhibition discovered in our study, cannot be corrected by targeting amyloid proteins alone. Our studies suggest that reducing the excessive GABA inhibition to the neurons in the brain’s dentate gyrus may lead to a novel therapy for Alzheimer’s disease. An ultimate successful therapy may be a cocktail of compounds acting on several drug targets simultaneously.’ Penn State University

An Ottawa-led team of researchers describe the role of a specific gene, called Snf2h, in the development of the cerebellum. Snf2h is required for the proper development of a healthy cerebellum, a master control centre in the brain for balance, fine motor control and complex physical movements.

Athletes and artists perform their extraordinary feats relying on the cerebellum. As well, the cerebellum is critical for the everyday tasks and activities that we perform, such as walking, eating and driving a car. By removing Snf2h, researchers found that the cerebellum was smaller than normal, and balance and refined movements were compromised.

Led by Dr. David Picketts, a senior scientist at the Ottawa Hospital Research Institute and professor in the Faculty of Medicine at the University of Ottawa, the team describes the Snf2h gene, which is found in our brain’s neural stem cells and functions as a master regulator. When they removed this gene early on in a mouse’s development, its cerebellum only grew to one-third the normal size. It also had difficulty walking, balancing and coordinating its movements, something called cerebellar ataxia that is a component of many neurodegenerative diseases.

‘As these cerebellar stem cells divide, on their journey toward becoming specialized neurons, this master gene is responsible for deciding which genes are turned on and which genes are packed tightly away,’ said Dr. Picketts. ‘Without Snf2h there to keep things organized, genes that should be packed away are left turned on, while other genes are not properly activated. This disorganization within the cell’s nucleus results in a neuron that doesn’t perform very well—like a car running on five cylinders instead of six.’

The cerebellum contains roughly half the neurons found in the brain. It also develops in response to external stimuli. So, as we practice tasks, certain genes or groups of genes are turned on and off, which strengthens these circuits and helps to stabilize or perfect the task being undertaken. The researchers found that the Snf2h gene orchestrates this complex and ongoing process. These master genes, which adapt to external cues to adjust the genes they turn on and off, are known as epigenetic regulators.

‘These epigenetic regulators are known to affect memory, behaviour and learning,’ said Dr. Picketts. ‘Without Snf2h, not enough cerebellar neurons are produced, and the ones that are produced do not respond and adapt as well to external signals. They also show a progressively disorganized gene expression profile that results in cerebellar ataxia and the premature death of the animal.’

There are no studies showing a direct link between Snf2h mutations and diseases with cerebellar ataxia, but Dr. Picketts added that it ‘is certainly possible and an interesting avenue to explore.’

In 2012, Developmental Cell published a paper by Dr. Picketts’ team showing that mice lacking the sister gene Snf2l were completely normal, but had larger brains, more cells in all areas of the brain and more actively dividing brain stem cells. The balance between Snf2l and Snf2h gene activity is necessary for controlling brain size and for establishing the proper gene expression profiles that underlie the function of neurons in different regions, including the cerebellum. Ottawa Hospital Research Institute