IBioBA scientists identify a circular RNA as a new regulator of the stress response

In a study published today in the prestigious journal Science Advances, an IBioBA team was able to shed light on the role that a type of circular RNA, which is abundant in the nervous system but so far unexplored, plays in emotional behaviour.

A job interview, an exam, a public lecture, or just everyday thoughts can provoke anxiety – a normal emotion of unease or agitation that is triggered in anticipation of stressful stimuli. Anxiety involves the expectation of a potential future threat and triggers the adoption of physiological changes that will adapt the body to cope with that threat.

However, in many cases, this normal, adaptive emotion gives way to a pathological state that is generally characterised by a response that is disproportionate in intensity or duration to situations that are often only subjectively perceived as threatening. According to the World Health Organization, anxiety disorders are the most common mental health conditions worldwide: in 2019 they affected more than 300 million people and, in the first year of the COVID-19 pandemic, global prevalence increased by 25%, exacerbating many mental health-related factors.

The Molecular Neurobiology group at IBioBA has been working for many years to unravel the neurobiological basis of stress-related behavioural disorders such as anxiety and depression. Despite the importance of this problem at a global level, and the great deal of research that has been done in this field, the molecular mechanisms that give rise to these disorders are still unknown. For this reason, the laboratory led by Damián Refojo is looking for possible answers by studying molecules and cellular mechanisms that have not yet been explored.

This is the context of the research published today in Science Advances, in which the group managed to functionally characterize a specific subtype of RNA called ‘circular’ that is especially abundant in neurons and is present in neuronal synapses: circTulp4, a molecule that was virtually unknown and which, from this work, is known to function as a promoter of synaptic neurotransmission, i.e. the basis of communication between neurons.

Initial studies were carried out in neuronal cultures, but to understand what the relevance of this circular RNA on behaviour might be, the group used the gene-editing technique called CRISPR-Cas9 to modify the genome of a mouse to eliminate the presence of circTulp4 from its brain. By studying the behaviour of these mice, they observed that the absence of circTulp4 increased sensitivity to aversive or stressful stimuli, suggesting that this molecule plays an inhibitory role on the circuits that control the stress response.

“When we talk about sensitivity to aversive stimuli, we refer to how easily a person or an organism can feel or perceive unpleasant, painful or simply threatening events or situations. There are molecules that regulate the brain’s sensing circuits to detect these kinds of potentially harmful or dangerous signals”, explains Sebastián Giusti, CONICET researcher and first author of the paper. CircTulp4 regulates the circuits that tune the response to the intensity of the environmental stimulus: “There are stimuli that become potentially dangerous, so it is adaptive in animals to implement stress-response behaviours when the intensity of those stimuli has exceeded a certain threshold; on the contrary, it is not adaptive to react when they are below that threshold”, he adds. It is worth noting that chronic alterations in these stress control mechanisms are often present or precede the onset of psychiatric illnesses such as depression or anxiety.

The little-known circTulp4

Circular RNAs (circRNAs), a type of non-coding RNA, i.e. RNAs that do not produce proteins (one of the main functions of RNA in cells), but perform functions per se, were discovered more than three decades ago. However, their functional significance remained enigmatic until recent advances in sequencing technologies shed light on their abundance and possible functions.

In 2015, the group participated in a collaborative study that produced the first molecular catalogue of circular RNAs present in different brain areas, in specific types of neurons and even within synaptic compartments. “It was then that we knew that this new class of RNA was very relevant in the nervous system, but we didn’t know anything about its function”, says Damian Refojo, researcher and director of IBioBA.

In the following years, a few working groups described some effects of circular RNAs in neurons, but to study the functions on complex behaviours, animal models were needed, and obtaining these models involves complex and expensive genetic engineering techniques. This is why there was a significant lack of scientific work showing whether these circular RNAs could indeed play a role in complex behaviours, such as memory, learning or emotional behaviour.

Intrigued, then, by the abundance of circular RNAs in the nervous system, the research team focused on circTulp4 to study its functional impact. “There is a lot of evidence suggesting that different mental disorders have their origin in abnormal synapse functioning, and circTulp4 is not only one of the most abundant circular RNAs in the brain, but also one of the most abundant in the synapse itself”, explains Giusti.

Using genetic engineering techniques, they created a circTulp4-deficient mouse model that allowed them to investigate in detail its real importance on different aspects of behaviour. To do this, they performed an extensive neurological and behavioural assessment focusing on stress-related behaviours, anxiety and depression. The team observed that in principle this molecule affects behaviour in stressful or anxiogenic situations. “Our results indicate that we would be in the presence of a new type of endogenous anti-stress molecule and probably even anxiolytic”, says Refojo.

Implications and future perspectives

In the future, the lab intends to investigate in depth the molecular mechanisms underlying the cellular and behavioural effects of circTulp4. “To better understand how circTulp4 acts, we need to continue with more in-depth biochemical and molecular studies, and that is what we have already started to do. In addition, it is important to extend the studies to new circular RNAs that we believe, based on preliminary data, may also be relevant in the functioning of the central nervous system”, says Giusti.

Understanding the function of circTulp4 represents an important finding in deciphering the function of circular RNAs in the nervous system. “These findings underline the broad potential of circular RNAs as relevant players not only in neuronal function, but also in the regulation of complex behaviours and probably also in the understanding of psychiatric disorders whose pathological basis is still poorly understood, which prevents us from having better therapeutic tools. Moreover, the COVID pandemic has left us with a transcendent legacy and a transcendent lesson. The legacy, RNA-based therapies, a new therapeutic strategy that is here to stay. The lesson: only those who master these technologies will be in a position to develop new therapies or address new challenges in human or animal health”, concludes Refojo.