Ravi Manjithaya, an autophagy researcher, talks about how the celluar housekeeping system that gets rid of toxins is not only a line of defence for the body but holds clues to understanding disease and ageing.

BY GBSNP VARMA

Proteins are the workhorses that build the structures for life, cell by cell, tissue by tissue, organ by organ. They’re the molecules that drive all biological processes, working tirelessly to keep cells healthy and functioning. As with everything, proteins too wear out, thousands of them every minute—becoming functionless; they misfold; they go bad. Cells make new proteins, even as they clean up the old ones, and on and on it goes.

Without the auto house-cleaning mechanism within the cell itself—autophagy or “self-eating” in Greek—cellular trash can turn toxic and eventually kill the cell.  Disease follows and ultimately death occurs.

In the last few years, researchers started believing that a misfiring autophagy system leads to a range of diseases, including infections, diabetes, cancer, and neurodegenerative disorders such as Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis and so on.

Autophagy recycles the degraded stuff as well, providing energy to the cell. When a cell doesn’t get energy without food, it banks on autophagy to provide energy. In fact, autophagy is considered an evolutionary response to starvation. It is the cell’s ability to capture, degrade and recycle anything inside itself.

For discovering and experimenting on autophagy Japanese biologist Yoshinori Ohsumi was awarded the Nobel Prize for medicine last year. Many labs around the world are working to understand the full range of possibilities in this field.

Ravi Manjithaya’s lab at Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bengaluru, is the first in India to focus directly on how autophagy happens at the basic level. Although a lot of information about autophagy exists, there is not much on how fast or slow—the speed, the rate—it happens. Also, there are many steps between capturing the trash and degrading it and recycling it. The lab is working to understand them. Using chemical biology approach—using small molecules to understand the biological processes, Manjithaya and his colleagues tested using high throughput screening approach about 1,500 compounds on protein clumps that cause Parkinson’s and found a couple potentially promising. Their work appeared recently in peer-reviewed journal Autophagy.

Manjithaya, trained first in molecular biology and later, in cell biology, has always been enthralled by the microscopic world of cell and its intricate workings. To look under microscope and find an unseen world, is amazing, he feels.

Excerpts from an interview:

Tell us about your fascination with cells and looking under the microscope.

Whether it was looking at microscopic creatures from the pond water or fluorescently labelled proteins and organelles such as peroxisomes and mitochondria, microscopy reveals a surreal, invisible world that never ceases to surprise and intrigue me. And now with new technologies breaking the diffraction barrier (resolution limit of light) and combining them with other emergent technologies, one gets to peep into the microscopic world with unprecedented clarity.

Interesting things you learned along the way.

Autophagy can gobble everything inside a cell…well, almost!

How did your doctoral and post doctoral studies help you feed into your current focus?

In my PhD, my supervisor, Prof. Rajan Dighe, taught me the importance of good assays, these are the key experimental tools. Towards the end of my PhD, I met Prof. Suresh Subramani who made me an offered of work in an emerging field called autophagy. His lab was an amalgam of people from diverse countries and his keen listening ability and persistent questions hooked me to autophagy research. Post doc work introduced me to various aspects of cell biology and the fact that there can be common themes running from the humble yeast to complicated human cells, caught my attention.

You say in postdoc you learnt some ‘tricks’, could you talk about them?

I learnt that cells can build up enormous amounts of a cellular compartment (organelle) dealing with fat metabolism—peroxisomes during certain conditions and then completely self-devour them under different situations. I used this idea to design an assay for a chemical biology approach to study autophagy.

How did the tricks you learnt help you apply here, in your lab.

Using the assay, my lab could identify new small molecules that affected autophagy in various ways. By studying how these molecules affect autophagy, we learnt new things about how cells control autophagy—either slow it down or massively increase it.

How does autophagy affect human health and wellbeing?

Basically, with autophagy, cells eventually become unhealthy due to accumulating garbage inside them and they die. Thus, neurons die if they don’t remove the protein clumps as in neurodegenerative disorders such as Parkinson’s, and Alzheimer’s. Similarly, in infectious diseases, when microorganisms enter our cells they encounter autophagy as a line of defence. Finally, it is believed that several aggressively growing cancer cells rely on autophagy to provide them with nutrients internally when exogenous nutrient supply is insufficient to fuel their hyper growth metabolism.

What’s the mechanism?

Although there are different modes of autophagy, the well-studied one is macroautophagy. It is more akin to a Pac-Man or a vacuum cleaner that seeks, captures intracellular material in a bubble-wrap like structure (autophagosomes) and delivers them to the lysosome (suicide bags) to degrade and recycle the captured contents.

The tentacles of disease reach far. What role does autophagy play here?

Efficient house cleaning is important for a healthy home. Similarly, autophagy keeps cells tidy and is a form of quality control to check and clear cellular components that may be in excess or dysfunctional. For example, proteins and other cellular materials such as parts of mitochondria wear out and must be safely removed and their parts recycled. Of the many different ways cells take care of trash, autophagy is considered a major pathway. Therefore, it is not surprising that breakdown of autophagy has been implicated in a vast number of diseases.

Why is the autophagy work important?

From the basic question about how autophagy is regulated—turned on or turned off—to the diverse number of disease states it impinges on, modulating autophagy levels has been shown to have therapeutic potential. In fact increased autophagy has even been linked to a longer life span.

Where is that field going?

Work is moving towards discovering new cellular components that play a role in carrying out autophagy. Eventually scientists would like to have a handle on controlling autophagy at will and that too in specific cell types in our body that is affected by disease. And I think small molecules that regulate the rate of autophagy will show the way.

Could you talk about your recent paper?

In neurodegenerative diseases such as Parkinson’s, from an autophagy perspective, there are two problems. One, toxic protein aggregates build-up inside brain cells, and two, autophagy in these cells is inefficient to clear them. Autophagy is slow perhaps because they deliver the protein aggregates for degradation at a slower pace than the rate at which the aggregates are produced. Using our indigenous assay, we identified a small molecule, 6-Bio, that speeds up autophagy resulting in reduced protein aggregates and saving the neurons from certain death. This was shown in a preclinical mouse model of Parkinson’s. Thus, we show that small molecules that accelerate autophagy especially at the cargo delivery step have potential therapeutic value.

(Preclinical: one of the final stage proof-of-principle laboratory level experiments that, if promising, can then be taken up for drug property testing for clinical trials.)

How did you go about the research?

Because autophagy works in yeast, mice and humans, we used the yeast system as it is easy to work with, especially when working with thousands of small molecules. We tested these molecules for their ability to rescue yeast cells that were destined to die because of protein clumps similar to the ones seen in Parkinson’s. Many compounds failed but the ones that succeeded showed that they worked through autophagy to clear the toxic protein clumps. These compounds were further explored in human cells to do the same. Finally, the most promising ones were tested in a preclinical mouse model of Parkinson’s. In these mice, we looked for the ability of this molecule to enter the brain, induce autophagy, decrease protein aggregates and loss of physical attributes common to the disease.

Yeast, mouse, human cell lines—how did these work?

They all do autophagy. We employed yeast for the ease of screening compounds, human cells for testing the potency of the compounds and to elucidate their mechanism and finally, the mouse model to see if the compound works in an organism at the brain level.

Challenges, surprises, along the way.

As ours is a new lab, everything had to be started from scratch, so it was a steep learning curve. Establishing the mouse model tested our resilience and patience. But the way 6-Bio compound increased autophagy was surreal. It took a while to convince ourselves about the results we were looking at.

Could you talk about the particular molecule 6-Bio, its characteristics, what it does and how?

We think that the protein GSK3β is somehow associated with a braking mechanism that slows down autophagy. The molecule 6-Bio disengages GSK3β from the autophagy machinery, thus speeding up the process enormously. Using a complementary genetic engineering approach to remove GSK3β also gave similar results in absence of 6-Bio, suggesting that GSK3β indeed slows down autophagy progression. 6-Bio as a drug still has issues associated with potential drug-like molecules such as solubility, toxicity, etc., which have to be worked on.

Finding a new cellular pathway; explain, please.

GSK3β involvement in autophagy has been well documented. We observed that it also participated in slowing down the delivery of protein aggregates for degradation. This so-called braking mechanism as we call it, we think, if targeted by small molecules such as 6-Bio, holds the key to increase autophagy and clearance of bad protein clumps. This, therefore, has therapeutic implications in neurodegenerative disorders.

To test the drug molecule in humans, what will be challenges.

From mouse to humans, a drug may take several years. The costs involved are huge, the compound clearly has issues with respect to safety, solubility, long term usage—basically it has to go through the whole 9-yards. Most importantly, patent related issues also have to be looked into. We have an Intellectual Property Cell at JNCASR which is helping us patent the process by which we identify these small molecules and the compounds that we have identified that have potential therapeutic application in diseases such as neurodegeneration, infection and cancer. The IP process is  long and cumbersome, not  mention the expenses involved. And is very different from writing a manuscript, but having the IP cell helps ease things out to an extent.

Autophagy working in the body, as compared with working  in brain. What are the intricacies here? How does autophagy work in different areas?

Studies shown that the vigour with which autophagy is carried out by cells all over our body is not the same. The reasons are not clearly known. But in terms of manipulating autophagy by external cues such as starvation, it seems that the brain is the final frontier. While many body tissues and organs respond to autophagy, in the brain it is sluggish at the best and it is not easily regulated. Finding these answers will be key in realising the therapeutic potential of autophagy in the brain and other organs in our body.

Neurodegenerative diseases are genetic in some people. When you don’t have that, is it lifestyle-related? How does lifestyle affect the cleaning-up of garbage, in what ways does it lead to pile up?

Although many genes have been identified that are responsible for neurodegenerative disorders, majority of the cases are believed to be not due to genetic reasons. Active lifestyle and healthy eating and resting habits have an impact on cellular health and on the body in general. Autophagy function also declines with age and a sedentary lifestyle is not clearly going help your neurons and other body cells to get rid of cellular trash and maintain quality control.

For people with Parkinson’s and others, there are boxing programmes in the West. There are intense, gruelling exercise programmes. People seem to do well.

What’s your take on that, given that it’s extremely difficult to study autophagy in humans.

Well, there are several high-profile studies showing in mice and other model systems, that if they are put on a good exercise routine and calorie-restricted diet, they live longer and are healthier.

Could you please talk about people—doctors, clinicians—working on neurodegnerative disorders, and what have you learned from them?

I speak often with a doctor, Nagashayana Natesh, who treats such patients. He laments the absence of both: unavailability of early diagnostic tools and no cure being available. Learning about the suffering of the patients and helplessness of the doctors who cannot offer a cure but provide only symptomatic relief to myriads of problems in these patients is what motivates us further to do research.

What could we do about these problems, at the scientific, clinical, policy levels?

Early detection is the key. And then setting up an active life style with constant engagement with these patients may slow down the disease. However, the elderly are getting increasingly isolated in our society.

Could you talk about the thrill of doing basic science?

It is for me a chance to become Sherlock Holmes. We start with a problem and a hypothesis. Accumulating data from lots of experiments (many failed ones) puts things in perspective and hopefully a potential solution to the problem emerges. This journey is unique, in a different world, and immensely satisfying but difficult to share this excitement.

Translational angle. Potential applications. What do you think about it?

We don’t worry about it. Discoveries in basic science eventually reflect surprising translational potential. For example, our intention is to use small molecules as tools to understand autophagy, the payoff could be a molecule like 6-Bio that shows translational potential.

What does it take to do more basic science?

Good scientific problems, enthusiastic students, courage, passionate collaborators, a place like JNCASR, and yes, if promised funds arrived on time, it would be great.

(GBSNP Varma is a freelance journalist based in Andhra Pradesh.)

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