Kovalev, EIPOD Postdoctoral Fellow at EMBL Hamburg’s Schneider Group and EMBL-EBI’s Bateman Group, is a physicist passionate about solving biological problems. He is particularly hooked by rhodopsins, a group of colorful proteins that enable aquatic microorganisms to harness sunlight for energy.

“In my work, I search for unusual rhodopsins and try to understand what they do,” said Kovalev. “Such molecules could have undiscovered functions that we could benefit from.”

Some rhodopsins have already been modified to serve as light-operated switches for electrical activity in cells. This technique, called optogenetics, is used by neuroscientists to selectively control neuronal activity during experiments. Rhodopsins with other abilities, such as enzymatic activity, could be used to control chemical reactions with light, for example.

Having studied rhodopsins for years, Kovalev thought he knew them inside out – until he discovered a new, obscure group of rhodopsins that were unlike anything he had seen before.

As it often happens in science, it started serendipitously. While browsing online protein databases, Kovalev spotted an unusual feature common to microbial rhodopsins found exclusively in very cold environments, such as glaciers and high mountains. “That’s weird,” he thought. After all, rhodopsins are something you typically find in seas and lakes.

These cold-climate rhodopsins were almost identical to each other, even though they evolved thousands of kilometres apart. This couldn’t be a coincidence. They must be essential for surviving in the cold, concluded Kovalev, and to acknowledge this, he named them ‘cryorhodopsins’.

Rhodopsins out of the blue

Kovalev wanted to know more: what these rhodopsins look like, how they work, and, in particular, what color they are.

Color is the key feature of each rhodopsin. Most are pink-orange – they reflect pink and orange light, and absorb green and blue light, which activates them. Scientists strive to create a palette of different colored rhodopsins, so they could control neuronal activity with more precision. Blue rhodopsins have been especially sought-after because they are activated by red light, which penetrates tissues more deeply and non-invasively.

To Kovalev’s amazement, the cryorhodopsins he examined in the lab revealed an unexpected diversity of colors, and, most importantly, some were blue.

The color of each rhodopsin is determined by its molecular structure, which dictates the wavelengths of light it absorbs and reflects. Any changes in this structure can alter the color.

“I can actually tell what’s going on with cryorhodopsin simply by looking at its color,” laughed Kovalev.

Applying advanced structural biology techniques, he figured out that the secret to the blue color is the same rare structural feature that he originally spotted in the protein databases.

“Now that we understand what makes them blue, we can design synthetic blue rhodopsins tailored to different applications,” said Kovalev.

Next, Kovalev’s collaborators examined cryorhodopsins in cultured brain cells. When cells expressing cryorhodopsins were exposed to UV light, it induced electric currents inside them. Interestingly, if the researchers illuminated the cells right afterwards with green light, the cells became more excitable, whereas if they used UV/red light instead, it reduced the cells’ excitability.

“New optogenetic tools to efficiently switch the cell’s electric activity both ‘on’ and ‘off’ would be incredibly useful in research, biotechnology and medicine,” said Tobias Moser, Group Leader at the University Medical Center Göttingen who participated in the study. “For example, in my group, we develop new optical cochlear implants for patients that can optogenetically restore hearing in patients. Developing the utility of such a multi-purpose rhodopsin for future applications is an important task for the next studies.”

“Our cryorhodopsins aren’t ready to be used as tools yet, but they’re an excellent prototype. They have all the key features that, based on our findings, could be engineered to become more effective for optogenetics,” said Kovalev.

Evolution’s UV light protector

When exposed to sunlight even on a rainy winter day in Hamburg, cryorhodopsins can sense UV light, as shown using advanced spectroscopy by Kovalev’s collaborators from Goethe University Frankfurt led by Josef Wachtveitl. Wachtveitl’s team showed that cryorhodopsins are in fact the slowest among all rhodopsins in their response to light. This made the scientists suspect that those cryorhodopsins might act like photosensors letting the microbes ‘see’ UV light – a property unheard of among other cryorhodopsins.

“Can they really do that?” Kovalev kept asking himself. A typical sensor protein teams up with a messenger molecule that passes information from the cell membrane to the cell’s inside.

Kovalev grew more convinced, when together with his collaborators from Alicante, Spain, and his EIPOD co-supervisor, Alex Bateman from EMBL-EBI, they noticed that the cryorhodopsin gene is always accompanied by a gene encoding a tiny protein of unknown function – likely inherited together, and possibly functionally linked.

Kovalev wondered if this might be the missing messenger. Using the AI tool AlphaFold, the team were able to show that five copies of the small protein would form a ring and interact with the cryorhodopsin. According to their predictions, the small protein sits poised against the cryorhodopsin inside the cell. They believe that when cryorhodopsin detects UV light, the small protein could depart to carry this information into the cell.

“It was fascinating to uncover a new mechanism via which the light-sensitive signal from cryorhodopsins could be passed on to other parts of the cell. It is always a thrill to learn what the functions are for uncharacterised proteins. In fact, we find these proteins also in organisms that do not contain cryorhodopsin, perhaps hinting at a much wider range of jobs for these proteins.”

Why cryorhodopsins evolved their astonishing dual function – and why only in cold environments – remains a mystery.

“We suspect that cryorhodopsins evolved their unique features not because of the cold, but rather to let microbes sense UV light, which can be harmful to them,” said Kovalev. “In cold environments, such as the top of a mountain, bacteria face intense UV radiation. Cryorhodopsins might help them sense it, so they could protect themselves. This hypothesis aligns well with our findings.”

“Discovering extraordinary molecules like these wouldn’t be possible without scientific expeditions to often remote locations, to study the adaptations of the organisms living there,” added Kovalev. “We can learn so much from that!”

Unique approach to unique molecules

To reveal the fascinating biology of cryorhodopsins, Kovalev and his collaborators had to overcome several technical challenges.

One was that cryorhodopsins are nearly identical in structure, and even a slight change in the position of a single atom can result in different properties. Studying molecules at this level of detail requires going beyond standard experimental methods. Kovalev applied a 4D structural biology approach, combining X-ray crystallography at EMBL Hamburg beamline P14 and cryo-electron microscopy (cryo-EM) in the group of Albert Guskov in Groningen, Netherlands, with protein activation by light.

“I actually chose to do my postdoc at EMBL Hamburg, because of the unique beamline setup that made my project possible,” said Kovalev. “The whole P14 beamline team worked together to tailor the setup to my experiments – I’m very grateful for their help.”

Another challenge was that cryorhodopsins are extremely sensitive to light. For this reason, Kovalev’s collaborators had to learn to work with the samples in almost complete darkness.

The genetic mutation causes an enzyme called leucine-rich repeat kinase 2, or LRRK2, to be overactive. Too much LRRK2 enzyme activity changes the structure of brain cells in a way that disrupts crucial communication between neurons that make the neurotransmitter dopamine and cells in the striatum, a region deep in the brain that is part of the dopamine system and is involved in movement, motivation and decision making.

“Findings from this study suggest that inhibiting the LRRK2 enzyme could stabilize the progression of symptoms if patients can be identified early enough,” said Suzanne Pfeffer, PhD, the Emma Pfeiffer Merner Professor in Medical Sciences and a professor of biochemistry. Researchers can mitigate overactive LRRK2 using MLi-2 LRRK2 kinase inhibitor, a molecule that attaches to the enzyme and decreases its activity.

Pfeffer added that because the genetic mutation is not the only way to end up with overactive LRRK2 enzyme, the inhibitor treatment might help with other types of Parkinson’s disease or even other neurodegenerative diseases.

Pfeffer is the senior author of the study published in Science Signaling on July 1. Ebsy Jaimon, PhD, a postdoctoral scholar in biochemistry, is the lead author. The work is part of a longstanding collaboration with Dario Alessi, PhD, at the University of Dundee in Scotland.

Cellular antennae

About 25% of Parkinson’s disease cases are caused by genetic mutations, and the single genetic mutation that makes the LRRK2 enzyme too active is one of the most common. An overactive LRRK2 enzyme causes cells to lose their primary cilia, a cellular appendage that acts like an antenna, sending and receiving chemical messages. A cell that has lost its primary cilia is like your mobile phone when the network is down — no messages come through or are sent.

In a healthy brain, many messages are sent back and forth between dopamine neurons in a region of the brain called the substantia nigra and the striatum. These cellular “conversations” are possible because dopamine neuron axons, which are tubular extensions coming off the cell body, reach all the way to the striatum to communicate with neurons and glia, cells that support neuronal function.

An important communication that is disrupted by too much LRRK2 enzyme activity occurs when dopamine neurons are stressed and release a signal in the striatum called sonic hedgehog (named after the cartoon character). In a healthy brain, it causes certain neurons and astrocytes, a type of glial support cell, in the striatum to produce proteins called neuroprotective factors. As their name suggests, these proteins help shield other cells from dying. When there is too much LRRK2 enzyme activity, many of the striatal cells lose their primary cilia — and their ability to receive the signal from dopamine neurons. This disruption in sonic hedgehog signaling means that needed neuroprotective factors are not produced.

“Many kinds of processes necessary for cells to survive are regulated through cilia sending and receiving signals. The cells in the striatum that secrete neuroprotective factors in response to hedgehog signals also need hedgehog to survive. We think that when cells have lost their cilia, they are also on the pathway to death because they need cilia to receive signals that keep them alive,” Pfeffer explained.

Restored cilia were unexpected

The goal of the study was to test if the MLi-2 LRRK2 kinase inhibitor reversed the effects of too much LRRK2 enzyme activity. Because the neurons and glia that were examined in this study were fully mature and no longer reproducing through cell division, the researchers were initially unsure whether cilia could regrow. Working with mice with the genetic mutation that causes overactive LRRK2 and symptoms consistent with early Parkinson’s disease, the scientists first tried feeding the mice the inhibitor for two weeks. There were no changes detected in brain structure, signaling or the viability of the dopamine neurons.

Recent findings on neurons involved in regulating circadian rhythms, or sleep-wake cycles, inspired the researchers to try again. The primary cilia on those cells — which were also no longer dividing — grew and shrank every 12 hours.

“The findings that other non-dividing cells grow cilia made us realize that it was theoretically possible for the inhibitor to work,” Pfeffer said.

The team decided to see what happened after mice with overactive LRRK2 enzyme consumed the inhibitor for a longer period of time; Pfeffer described the results as “astounding.”

After three months of eating the inhibitor, the percentage of striatal neurons and glia typically affected by the overactive LRRK2 enzyme that had primary cilia in mice with the genetic mutation was indistinguishable from that in mice without the genetic mutation. In the same way moving from an area with spotty cell service to one with good service restores our ability to send and receive text messages, the increase in primary cilia restored communication between dopamine neurons and the striatum.

The striatal neurons and glia were again secreting neuroprotective factors in response to hedgehog signaling from dopamine neurons in the same amounts as the brains of mice without the genetic mutation. The hedgehog signaling from dopamine neurons decreased, suggesting they were under less stress. And, indicators of the density of dopamine nerve endings within the striatum doubled, suggesting an initial recovery for neurons that had been in the process of dying.

“These findings suggest that it might be possible to improve, not just stabilize, the condition of patients with Parkinson’s disease,” Pfeffer said.

The earliest symptoms of Parkinson’s disease begin about 15 years before someone notices a tremor. Typically, these symptoms are a loss of smell, constipation and a sleep disorder in which people act out their dreams while still sleeping, according to Pfeffer. She said the hope is that people who have the LRRK2 genetic mutation can start a treatment that inhibits the enzyme as early as possible.

The next step for the research team is to test whether other forms of Parkinson’s disease that are not associated with the LRRK2 genetic mutation could benefit from this type of treatment.

“We are so excited about these findings. They suggest this approach has great promise to help patients in terms of restoring neuronal activity in this brain circuit,” Pfeffer said. “There are multiple LRRK2 inhibitor clinical trials underway, and our hope is that these findings in mice will hold true for patients in the future.”

The study was funded by The Michael J. Fox Foundation for Parkinson’s Research, the Aligning Science Across Parkinson’s initiative and the United Kingdom Medical Research Council.

The linchpin is the system’s ability to analyze long-underused heart imaging, alongside a full spectrum of medical records, to reveal previously hidden information about a patient’s heart health.

The federally-funded work, led by Johns Hopkins University researchers, could save many lives and also spare many people unnecessary medical interventions, including the implantation of unneeded defibrillators.

“Currently we have patients dying in the prime of their life because they aren’t protected and others who are putting up with defibrillators for the rest of their lives with no benefit,” said senior author Natalia Trayanova, a researcher focused on using artificial intelligence in cardiology. “We have the ability to predict with very high accuracy whether a patient is at very high risk for sudden cardiac death or not.”

The findings are published today in Nature Cardiovascular Research.

Hypertrophic cardiomyopathy is one of the most common inherited heart diseases, affecting one in every 200 to 500 individuals worldwide, and is a leading cause of sudden cardiac death in young people and athletes.

Many patients with hypertrophic cardiomyopathy will live normal lives, but a percentage are at significant increased risk for sudden cardiac death. It’s been nearly impossible for doctors to determine who those patients are.

Current clinical guidelines used by doctors across the United States and Europe to identify the patients most at risk for fatal heart attacks have about a 50% chance of identifying the right patients, “not much better than throwing dice,” Trayanova says.

The team’s model significantly outperformed clinical guidelines across all demographics.

Multimodal AI for ventricular Arrhythmia Risk Stratification (MAARS), predicts individual patients’ risk for sudden cardiac death by analyzing a variety of medical data and records, and, for the first time, exploring all the information contained in the contrast-enhanced MRI images of the patient’s heart.

People with hypertrophic cardiomyopathy develop fibrosis, or scarring, across their heart and it’s the scarring that elevates their risk of sudden cardiac death. While doctors haven’t been able to make sense of the raw MRI images, the AI model zeroed right in on the critical scarring patterns.

“People have not used deep learning on those images,” Trayanova said. “We are able to extract this hidden information in the images that is not usually accounted for.”

The team tested the model against real patients treated with the traditional clinical guidelines at Johns Hopkins Hospital and Sanger Heart & Vascular Institute in North Carolina.

Compared to the clinical guidelines that were accurate about half the time, the AI model was 89% accurate across all patients and, critically, 93% accurate for people 40 to 60 years old, the population among hypertrophic cardiomyopathy patients most at-risk for sudden cardiac death.

The AI model also can describe why patients are high risk so that doctors can tailor a medical plan to fit their specific needs.

“Our study demonstrates that the AI model significantly enhances our ability to predict those at highest risk compared to our current algorithms and thus has the power to transform clinical care,” says co-author Jonathan Crispin, a Johns Hopkins cardiologist.

In 2022, Trayanova’s team created a different multi-modal AI model that offered personalized survival assessment for patients with infarcts, predicting if and when someone would die of cardiac arrest.

The team plans to further test the new model on more patients and expand the new algorithm to use with other types of heart diseases, including cardiac sarcoidosis and arrhythmogenic right ventricular cardiomyopathy.

Authors include Changxin Lai, Minglang Yin, Eugene G. Kholmovski, Dan M. Popescu, Edem Binka, Stefan L. Zimmerman, Allison G. Hays, all of Johns Hopkins; Dai-Yin Luand M. Roselle Abrahamof the Hypertrophic Cardiomyopathy Center of Excellence at University of California San Francisco; and Erica Schererand Dermot M. Phelanof Atrium Health.

Cardiovascular disease is the leading cause of death worldwide. There is a large body of evidence linking poor air quality with cardiovascular disease. However, the underlying changes in the heart resulting from air pollution exposure are unclear.

“We know that if you’re exposed to air pollution, you’re at higher risk of cardiac disease, including higher risk of having a heart attack,” said the study’s senior author Kate Hanneman, M.D., M.P.H., from the Department of Medical Imaging at the Temerty Faculty of Medicine, University of Toronto and University Health Network in Toronto. “We wanted to understand what drives this increased risk at the tissue level.”

Dr. Hanneman and colleagues used cardiac MRI, a noninvasive imaging technique, to quantify myocardial fibrosis and assess its association with long-term exposure to particles known as PM_2.5. At 2.5 micrometers in diameter or less, PM_2.5 particles are small enough to enter the bloodstream through the lungs. Common sources include vehicle exhaust, industrial emissions and wildfire smoke.

The researchers wanted to evaluate the effects of air pollution on both healthy people and those with heart disease, so the study group included 201 healthy controls and 493 patients with dilated cardiomyopathy, a disease that makes it more difficult for the heart to pump blood.

Higher long-term exposure to fine particulate air pollution was linked with higher levels of myocardial fibrosis in both the patients with cardiomyopathy and the controls, suggesting that myocardial fibrosis may be an underlying mechanism by which air pollution leads to cardiovascular complications. The largest effects were seen in women, smokers and patients with hypertension.

The study adds to growing evidence that air pollution is a cardiovascular risk factor, contributing to residual risk not accounted for by conventional clinical predictors such as smoking or hypertension.

“Even modest increases in air pollution levels appear to have measurable effects on the heart,” Dr. Hanneman said. “Our study suggests that air quality may play a significant role in changes to heart structure, potentially setting the stage for future cardiovascular disease.”

Knowing a patient’s long-term air pollution exposure history could help refine heart disease risk assessment and address the health inequities that air pollution contributes to both in level of exposure and effect. For instance, Dr. Hanneman said, if an individual works outside in an area with poor air quality, healthcare providers could incorporate that exposure history into heart disease risk assessment.

The air pollution exposure levels of the patients in the study were below many of the global air quality guidelines, reinforcing that there are no safe exposure limits.

“Public health measures are needed to further reduce long-term air pollution exposure,” Dr. Hanneman said. “There have been improvements in air quality over the past decade, both in Canada and the United States, but we still have a long way to go.”

In addition to illuminating the links between air pollution and myocardial fibrosis, the study highlights the important role that radiologists will play in research and clinical developments going forward.

“Medical imaging can be used as a tool to understand environmental effects on a patient’s health,” Dr. Hanneman said. “As radiologists, we have a tremendous opportunity to use imaging to identify and quantify some of the health effects of environmental exposures in various organ systems.”

Not to be confused with hurricanes or cyclones, these convective storms tend to be short-lived but powerful, with tree-toppling winds and lightning. In a perspective paper in Ecology Letters, Gora and colleagues lay out the case for why such storms could be a major driving force behind the rising death toll of tropical trees. As they become more common in the warming tropics, thunderstorms are a growing threat to trees and the carbon they store.

“Tropical forests have massive effects on global climate. They’re like the lungs of the Earth, and we’re seeing trees in them dying at higher rates than in the past, and the composition of forests is changing, too,” said Gora. “That could be really problematic for the future of not just tropical forests, but for the planet.”

Understanding what’s causing the trends in tree death is critical to guiding decisions about which tree species to plant or conserve in a forest, so that forest managers can ensure forests continue thriving and storing carbon long into the future.

“Being in the forest during a tropical storm is unforgettable,” said coauthor Vanessa Rubio, a forest ecologist in Gora’s lab at Cary Institute. “As the storm quickly builds, the sky darkens, humidity changes drastically, and strong winds shake the trees. Then, thunder and lightning come. Leaves and branches fall to the ground, rain pours down, and your instinct is to get back to the field station as quickly as possible.”

Despite their obvious danger to people, storms had been overlooked and understudied as a potential culprit in tree mortality trends. But when the team reanalyzed data from previous studies on tropical forest carbon stocks, they found that storms were at least as good as drought and temperature in explaining the patterns of tree mortality and forest carbon storage.

“We were surprised to find that storms may be the largest single factor causing tree death in these forests, and they’re largely overlooked by research into carbon storage in the tropics,” said Gora. “Our estimates suggest that storms are responsible for 30 to 60% of tree mortality in the past, and that number must be increasing as storm activity increases by 5 to 25% each decade.”

The team also added storms to the largest plot-based study of forest biomass carbon dynamics to date. That study had previously concluded that when temperatures go above a certain threshold, tropical forests experience a fast decline in carbon stocks. “But when you add storms, that relationship goes away,” said Gora. “It basically shows that you have to include storms, or you might not get the answers right.”

Storms and droughts are not mutually exclusive, the scientists note — the same forests can experience both high storm activity and drought stress. They found high convective storm activity across the southern Amazon, where water stress is also high and patterns of change are among the most extreme.

“During my studies on threats to tropical forests, my professors, our textbooks, and even overall climate policy never mentioned small, convective storms as a potential source of forest mortality,” said coauthor Ian McGregor, a Cary Institute forest ecologist in Gora’s lab. “I don’t remember seeing them in global climate models used to inform climate policy. Given our findings, however, it’s clear we need a more thorough understanding of these storms to have more accurate climate models, and thus more effective policy.”

There are good reasons why scientists have overlooked storms until now. Temperature and water stress can be monitored with meteorological stations and readily connected to long-term forest plot data. It is much harder to detect storms and track their highly localized damage. Mortality caused by thunderstorms is not easily detected via satellite, and it’s not practical for researchers on foot to survey large forested areas frequently enough to pinpoint the damage caused by a specific storm.

Gigante, a project led by Gora and co-author Adriane Esquivel-Muelbert from the University of Birmingham, offers one way to overcome these challenges. The project combines a lightning location system, drone scouts, and on-the-ground experts to sample large areas of tropical forest frequently. With these tools, they are starting to quantify when, where, and why tropical trees are dying, and which species are most affected.

Understanding current and future threats to tropical forests is crucial to informing long-term conservation and restoration efforts.

“If we make decisions about which species to plant or conserve based on an incorrect understanding of what’s actually killing these trees and which species are most vulnerable, those forests won’t reach their full potential,” said Gora. Storms are most deadly to mature trees, so the consequences of misguided reforestation efforts might not be known until decades after the trees are planted.

“However,” Gora continued, “if we can build a more holistic picture of what’s driving forest change, we can be a lot more confident in guiding forest management practices for long-term sustainability.”

Authors

• Evan M. Gora — Cary Institute of Ecosystem Studies, Smithsonian Tropical Research Institute
• Ian R. McGregor — Cary Institute of Ecosystem Studies
• Helene C. Muller-Landau — Smithsonian Tropical Research Institute
• Jeffrey C. Burchfield — University of Alabama, Huntsville
• KC Cushman — Oak Ridge National Laboratory
• Vanessa E. Rubio — Cary Institute of Ecosystem Studies
• Gisele Biem Mori — National Institute for Amazon Research, Universidade do Estado de Mato Grosso
• Martin J. P. Sullivan — Manchester Metropolitan University
• Matthew W. Chmielewski — University of Louisville
• Adriane Esquivel-Muelbert — University of Birmingham

Funding was provided in part by the National Science Foundation (NSF) grants DEB-2213245 and DEB-2241507 to EMG, and NE/W003872/1 to MS and EMG. AE-M was further funded by the Royal Society Standard Grant RGS\R1\221115 ‘MegaFlora’, the UK Research and Innovation/Natural Environment Research Council (NERC) TreeScapes NE/V021346/1 ‘MEMBRA’, the NERC/NSF Gigante NE/Y003942/1, and the Foundation for Research on Biodiversity/Centre for the Synthesis and Analysis of Biodiversity ‘Syntreesys’.

Cary Institute of Ecosystem Studies is an independent nonprofit center for environmental research. Since 1983, our scientists have been investigating the complex interactions that govern the natural world and the impacts of climate change on these systems. Our findings lead to more effective resource management, policy actions, and environmental literacy. Staff are global experts in the ecology of: cities, disease, forests, and freshwater.