Surprising discovery in the brain may have important implications for drug development

Disease and treatment 27. dec 2022 3 min Professor Dimitrios Stamou Written by Kristian Sjøgren

A single enzyme energises the loading of all neurotransmitters into synaptic vesicles, which store the neurotransmitters that are released at the synapse. New research shows that this single enzyme switches on and off completely randomly – sometimes taking hour-long breaks between switching. The discovery provides new insight into the regulation of neurotransmitters and may have important implications for drug development.

A quite unremarkable enzyme in the brain has baffled scientists because it does not behave as the researchers had expected.

Vacuolar-type adenosine triphosphatases (V-ATPases) are electrogenic rotary mechanoenzymes that establish electrochemical proton gradients in many cell processes, including in the brain, in which the loading of neurotransmitters in neurons is energised by about one V-ATPase molecule per synaptic vesicle. The electrochemical proton gradient established subsequently energises the secondary transport of all neurotransmitters into synaptic vesicles.

The neurotransmitters enable all signalling within the brain and from the brain to the rest of the body. V-ATPases are therefore an absolutely key enzyme in all mammals. Without the enzyme, no signalling: no life.

Because V-ATPases are so essential for life, an initial presumption might be that they are permanently active, but new research indicates otherwise.

V-ATPases switch on and off randomly and even take hour-long breaks, during which the electrogenic rotary enzymes do not energise the loading of neurotransmitters into the synaptic vesicles, potentially affecting neuronal communication.

The discovery is both surprising and contributes novel insight into the brain and the activity of individual molecules, which may contribute to the development of new drugs targeting many diseases.

“Developing medicines requires knowing how a potential medicine affects the body’s molecules. We typically investigate this based on an average of many molecules but not on a single molecule. But whether you affect one enzyme that is switched on or off can be very important when you have an enzyme like V-ATPases that enables all signalling between the neurons of the brain,” explains a researcher behind the study, Dimitrios Stamou, Professor, Department of Chemistry, University of Copenhagen.

The research has been published in Nature.

Only one enzyme loader at work

The V-ATPases use the energy released during ATP hydrolysis to energise the loading of neurotransmitters and during the process they concentrate the signal molecules up to 100-fold.

The V-ATPases function like small soap bubbles that merge with the cell membrane at a given signal and release all the neurotransmitters from the synaptic vesicles into the synapses. This results in numerous activities led by the nervous system, such as movement and storing memories.

The new study shows that, in neurons, about one V-ATPase molecule loads all neurotransmitters into one synaptic vesicle, and Dimitrios Stamou says that having an important cell system so dependent on just one enzyme is unique, adding:

“If V-ATPases do not function, no neurotransmitters are loaded into the synaptic vesicles, and then the neurons cannot send signals. In this study, we also showed that, most surprisingly, the V-ATPases do not function continuously but take hour-long breaks. In fact, despite their importance, the enzymes are only active 40% of the time.”

Why are 40% of V-ATPases inactive?

Dimitrios Stamou says that the discovery raises many questions about the brain, including the question of why the V-ATPases only function part time and that no other mechanism takes over the role of loading neurotransmitters into the synaptic vesicles. The brain must have another way around this problem, but this is not well understood.

Further, the study shows that the synaptic vesicles constantly leak protons, which are part of their cellular fuel – equivalent to driving a car with a leaking fuel tank.

The discovery has graced the cover of Nature, with the headline “Pump, rest, leak, repeat” – referring to the endless cycle that includes the V-ATPases.

“This raises questions we intend to investigate. Why are 40% of the V-ATPases inactive and what are the implications of neurotransmitters not being loaded into the synaptic vesicles? Has the body developed ways around this problem and what are they? Or, alternatively, could the fraction of active enzymes in itself encode information about how to control the flow of neurotransmitters? We do not know, but our study indicates that this needs to be investigated further,” explains Dimitrios Stamou.

Novel investigation techniques required

Part of the extraordinary discovery is the technology the researchers used.

Previously, researchers could not measure the activity of a single transporter enzyme. Dimitrios Stamou had to develop this technology, described in 2016. A crucial bottleneck with that technology, however, was that the enzymes to be studied had to be purified and then examined in a new artificial environment. This was especially a problem for mammalian enzymes, which stopped working when removed from their natural environment.

Using their new method, researchers entirely circumvent enzyme purification because they now remove a single intact synaptic vesicle from mice and study it at room temperature, where it continues to function for up to several hours.

This also means that researchers can carry out experiments on V-ATPases for longer and can detect the pauses before starting to load neurotransmitters again.

“Purified V-ATPases could only be measured for a few minutes, which was not enough time to detect their constant switching on and off,” says Dimitrios Stamou.

May be important for drug screening

The discovery may turn out to extend beyond new insight into the brain.

V-ATPases are a potential target for drugs to combat neurodegenerative diseases and also cancer. But when drug developers test their drugs for effectiveness against V-ATPases, they examine the effectiveness based on an average of enzymes – 40% of which are inactive.

Dimitrios Stamou says that this may potentially miss the effects of a drug candidate on a specific active enzyme.

“You can ask how many drug screenings are subject to bias, because we miss effects that are hidden in the average. This applies not only to effects in the brain but everywhere in the body, where many proteins are isolated in very small numbers. We are slowly starting to develop methods that enable us to study these proteins individually and how to approach them in relation to drug development,” concludes Dimitrios Stamou.

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching” has been published in Nature. In 2017, the Novo Nordisk Foundation awarded a Challenge Programme grant to Dimitrios Stamou for the project Center for Geometrically Engineered Cellular Systems.

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