Scientists at the University of Valparaíso use the vinegar fly to study genes linked to autism


Autism spectrum disorder (ASD) or autism is a neuro-genetic disorder related to brain development. The diagnosis of ASD is based on behavioral tests, which complicate its diagnosis during pregnancy or at birth.

According to the WHO, about one in 100 children has autism, with a bias of four boys for one girl. In Chile, according to the 2011 Guide to Clinical Practice, Detection and Diagnosis for Autism Spectrum Disorders (ASD) by the Ministry of Health (MINSAL), there is no registry of ASD diagnoses in the country.

ASD is quite heterogeneous among people, and its characteristics can coexist with other neurological conditions, such as attention deficit hyperactivity disorder (>50%), mental retardation (>40%), anxiety or sleep and atypical (>90%) sensory processing and integration. Numerous genetic studies on people with ASD have made it possible to isolate hundreds of risk genes relevant to the monogenic (inheritance of a trait influenced by a single gene) and oligogenic (inheritance of a trait influenced by a few genes) forms of the disorder. However, a major challenge is determining how mutations in a particular neurodevelopmental gene trigger specific ASD behavioral traits.

For this reason, a group of researchers led by John Ewer and Angelina Palacios of the Interdisciplinary Center of Neurosciences of the University of Valparaíso (CINV) used the vinegar fly or Drosophila to study the role of the TRPY gene, homolog of the human gene TRPC6 (or potential 6-channel transient receiver with its name in English) in the aspect of the TEA.

The TRPC6 gene

Drosophila is a very powerful genetic model in scientific research because its entire genome is known and it has been famously used to study genes related to the circadian rhythm, research that won the Nobel Prize in Physiology or Medicine in 2017. Furthermore, Drosophila has been an important system for studying the consequences of genes related to cognitive and psychiatric disorders, including several genes associated with ASD.

The TRPC6 gene contains the information to build a voltage-independent calcium channel (allows calcium to enter the cell without the cell being activated by an electrical signal). A genetic alteration has been identified in a person with ASD. However, knowledge of the function of TRPC6 in the human brain is very poor, and the consequences of gene disruption on its neuronal function have only been studied in stem cells derived from human neuronal cells.

That is why the UV researchers wanted to investigate the role of this gene and exploited some highly stereotyped behaviors of the vinegar fly, such as courtship behavior, the ability to generate learning and memory, circadian rhythm, locomotor activity and the sleep homeostasis, to analyze the consequences of an alteration of the TRPY gene on brain functioning, in a living animal model.

I study

First, the researchers used courtship as a social behavior to analyze flies induced with a mutation in the TRPY gene to see if the male pays attention to the female and adjusts his behavior based on her responses to her advances. During courtship, the male first orients himself towards the female (looks for her), then touches her with his forepaw and follows her while producing a “courtship song” by vibrating one of his wings. Depending on the receptivity of the female and after engagement of the male, the pair take additional steps in the fixed courtship sequence, culminating in copulation. If the female is not receptive or the male is not fully engaged, any step in the sequence may be extended or the sequence terminated as completed if the female rejects male.

The researchers observed that mutant males for the gene under investigation initiated and terminated courtship much faster than control males. Also, they had fewer copulation attempts. But this reduced latency (the time interval observed between mating and the onset of courtship behavior) did not affect female attractiveness. When the mutants were fed hyperforin (a drug that initiates a physiological response similar to the protein produced by the TRPC6 gene) four days before testing, the mutant males had a similar latency in courtship behavior to control males.

On the other hand, control males (without mutations) took longer to initiate courtship when mating with TRPY mutant females. Also, not all males performed all courtship steps, such as the “courtship song”, similar courtship to controls was observed when the female was fed hyperforin for four days prior to testing.

These results suggest that the TRPY gene is involved in the regulation of courtship in males. Thus, mutant males express a greater urge to copulate, while in females the gene regulates courtship attractiveness.

hyperactivity

Second, the researchers focused on hyperactivity and anxiety-like behavior in humans, but in mutant flies. It is known that 50% of people with ASD have hyperactivity disorder or attention deficit disorder or anxiety. To measure these behaviors, the researchers analyzed the locomotor activity of the flies, as well as the flies’ preference for being in the center (more anxiety) versus the edges (less anxiety) of a space called an “arena.”

The scientists found that male and female TRPY mutant flies were hyperactive. This hyperactivity was reflected in the increase in the total distance traveled within the arena compared to control flies. Furthermore, these flies spent more time in the central area of ​​the arena than the controls, but spent as much time at the edges as in the center of the arena. However, locomotor activity similar to controls was observed when the mutant flies were fed hyperforin for four days prior to testing.

These results suggest that the TRPY gene is related to locomotor activity and its mutation causes hyperactivity in flies, but does not produce anxiety-like behaviors.

Then the CINV researchers investigated whether the absence of the TRPY gene affected learning and memory in the mutant flies. This is because it has been observed that people with ASD can have important differences in these traits compared to the neurotypical population. Therefore, in order to evaluate these processes, the scientists focused their attention on courtship behavior. This test is based on the fact that a mated female rejects the advances of the male and eventually the male stops courting her. This results in the male learning and thus reducing his further courtship of any other female. This behavior can be used to measure short- and long-term memory in male flies.

Using this test, the scientists found that TRPY mutant males displayed low levels of learning, as evidenced by the fact that they continued to follow females who rejected them. Additionally, they showed lower memory levels of not learning from previous rejection when remated with other females than control males. However, when the mutant males were fed hyperforin for four days before the trial, they recovered their learning levels, but not their long-term memory.

Finally, the researchers analyzed sleep and sleep homeostasis in the mutant flies. It has been observed that people with ASD can have sleep disturbances and as additional information, the person who has been observed to have a copy of the TRPC6 gene disrupted has been diagnosed with sleep problems. Therefore, the scientists measured the sleep duration of the mutant flies for 24 hours and found that the mutant males, but not the females, had shorter sleep durations, with higher frequencies of very short sleep episodes. Next, the researchers measured sleep homeostasis. To do this test, they deprived the flies of sleep the night before the test using two-second mechanical stimulation every ten seconds for twelve hours a night. Next, they measured sleep duration and frequency parameters. They found that mutant males made up for less lost sleep than control males. In mutant females, there was only a reduction in sleep recovery compared to control females. However, when the male and female mutants were fed hyperforin before testing, they were able to resume normal sleep. These sleep problems observed in the mutant flies were not related to circadian rhythm, as the mutants had a circadian rhythm similar to the circadian cycle of the controls.

In conclusion, the research team led by John Ewer and Angelina Palacios demonstrated that the mutant flies for the TPRY gene expressed behavioral traits similar to those of people with ASD, including those of the patient with TRPC6 gene disruption, such as difficulties in interactions social impairments, hyperactivity, impairments in learning, memory and sleep homeostasis. All these traits were rescued in the flies carrying the mutation by introducing the TRPY gene or feeding them the drug hyperforin. This could indicate that the features seen in the mutant flies are caused by a lack of the TRPY gene. Furthermore, they demonstrated that Drosophila is a good model for studying ASD-related genes and can be a facilitator for analyzing correlations between different traits seen in people with ASD and the genes they have.

Aside from the scientific context provided by this study, we can become aware of the percentage of people with ASD currently in the world. It is important to understand not only the difficulties they may have in living in a normal-typical society, but also the contributions they may make to that society. In other words, it is important to have a commitment to a real inclusion of people with ASD in their daily life, work and social environment.

Fountain: https://www.nature.com/articles/s41380-022-01555-1

Additional reading: Ministry of Health (MINSAL) Clinical Practice Guide, Early Detection and Diagnosis of Autism Spectrum Disorders (ASD) 2011: https://www.minsal.cl/portal/url/item/bd81e3a09ab6c3cee040010164012ac2.pdf

*This article comes from the agreement with the Interdisciplinary Center of Neurosciences of the University of Valparaíso.

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