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:
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.
things you learned along the way.
Autophagy can gobble everything inside a cell…well, almost!
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.
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.
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.
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.
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.
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.
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
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
(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.)
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.
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
you talk about the particular molecule 6-Bio, its characteristics, what it does
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.
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.
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.
working in the body, as compared with working
in brain. What are the intricacies here? How does autophagy work in
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.
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.
your take on that, given that it’s extremely difficult to study autophagy in
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.
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.
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.
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.
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.
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.