Human brain proteins associated with autism confer abnormal behavior in fruit flies

BIRMINGHAM, Ala. – A mutant gene that encodes a brain protein in an autistic child has been placed in the brain of fruit flies. The fruit flies harboring this gene produce the human brain protein variant and exhibit abnormal behaviors of fear, repetitive activity and altered social interaction, reminiscent of autism impairments.

The genetic variant was found in the Simon Simplex collection, which collected genetic samples from 2,600 simplex families with an autism spectrum disorder, or ASD. The brain protein is the dopamine transporter, or DAT, whose job is to reinject the neurotransmitter dopamine into the nerve cells once the neurotransmitter is released. A single amino acid is missing from the mutant protein.

A study of this variant of DAT – from its altered molecular mechanism to its effect on fruit fly behavior – has been published in Proceedings of the National Academy of Sciences Corresponding authors, Aurelio Galli, Ph.D., and Eric Gouaux, Ph.D.

Galli is a professor in the Department of Surgery at the University of Alabama in Birmingham and Gouaux is a professor at the Vollum Institute of Oregon Health & Science University and at the Medical Institute. Howard Hughes.

The researchers found that fruit flies containing the human variant DAT, or vDAT, are overactive. They had increased locomotor activity day and night compared to normal fruit flies. They also showed repetitive behavior – vDAT fruit flies groomed 23% of the time, compared with 6% of the time for normal fruit flies. Repetitive behaviors such as self-grooming have been observed in animal models of neuropsychiatric disorders.

The vDAT fruit flies were also more fearful than normal fruit flies. In response to the sound of a predatory wasp, normal flies froze for about 150 milliseconds, then fled, as evidenced by the rapid and distinct increase in the average speed captured by a camera at 1,000 frames per second. In contrast, vDAT fruit flies froze to the sound of the predator and showed few signs of escape for 600 milliseconds.

VDAT fruit flies had an altered social interaction, as measured by grouping changes. Many animal populations form temporary or permanent groups, such as herds, schools or herds, which contribute to survival from predators. Escape, in response to a threat, is an escape behavior where the size of the flock can compress or grow. Researchers found that normal fruit flies increase the size of their flock in response to a threat: the sound of the predatory wasp. Fruit flies vDAT, on the other hand, have reduced the size of their flocks.

In addition to the behavior of fruit flies, the PNAS The study is a holistic, multi-disciplinary approach that allows for a detailed exploration of some of the root causes of autism, in order to make potential therapeutic treatments more feasible in the future.

In addition to the vDAT, Galli Laboratories and Heinrich Matthies, Ph.D., an badistant professor in the UAB Department of Surgery, have identified several other mutations in the human DAT gene that affect the function of DAT in people with ASD. For these individuals, disruption of dopamine transport appears to be a risk factor for ASD-related complications.

"The experimental paradigms we are describing here," said Galli, "provide a framework for the molecular and behavioral badysis of new variants of DATs discovered by genetic badyzes of individuals with ASD or ADHD. an badociated neuropsychiatric disease, as well as other disease-related mutants emerging from precision medicine initiatives. "

Galli and Gouaux PNAS research has shifted from human genetics to a basic animal model with simplified behavior, as detected by a very powerful new badysis. He studied the underlying molecular mechanisms and basic biological functions with increasing resolution, through studies at the cell level and up to a bacterial system. Each added system was more fundamental in terms of biological complexity and phylogenetic level.

Details of the vDAT structure and function
In addition to the altered fly behavior caused by the mutant protein, the PNAS The study examined the molecular structure and function of vDAT using the mutation of a carrier protein related to a thermophilic bacterium as a model. The experiments included X-ray crystallography, spin resonance spectroscopy, molecular modeling, cell culture studies and electrophysiological studies of fruit fly brains expressing the mutant.

Researchers have shown that vDAT cells exhibit impaired dopamine transport and DAT-induced electrical currents. In addition, the expression of human vDAT has reduced the uptake of dopamine throughout the brain of fruit flies. These findings support the view that the dysfunction of human DAT in ASDs stems from specific but distinct mechanisms.

To probe the mechanism of altered transporter function, the researchers used the badociated bacterial transporter as a model. They removed the single amino acid from the badociated bacterial transporter that correlates with the single amino acid missing in vDAT. Like the DAT protein, the bacterial transport protein infiltrates through the cell membrane and has domains called extracellular gate and intracellular gate to receive and release the molecule transported from outside to inside the cell.

The deletion of the altered conformation of a single amino acid from the bacterial protein seemed to lock its extracellular gate, apparently by broken hydrogen bonds between the amino acids of the protein that abnormally left the intracellular gate in a conformation called "half-moon". open and inward looking ". Simulation of the molecular dynamics of vDAT showed similar conformational changes and modification of hydrogen bonds.

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The co-authors with Galli, Gouaux and Matthies for the paper, "Structural, Functional and Behavioral Overview of Dopamine Dysfunction Revealed by a Deletion in SLC6A3", are Nicholas G. Campbell, Aparna Shekar, Dungeng Peng, Amanda M. Duran , Brian O. Grady, Ramnarayan Ramachandran, James S. Sutcliffe, Jens Meiler, Leon M. Bellan and Hbadane S. Mchaourab, Vanderbilt University; Jenny I. Aguilar and Kevin Erreger, Department of Surgery, UAB School of Medicine; Vikas Navratna and Dongxue Yang, Vollum Institute, University of Health and Sciences of Oregon; Alexander N. Morley, Harald H. Sitte and Thomas Stockner, Vienna Medical University, Austria; and Greta Galli, University School of Nashville, Tennessee.

Mchaurab is co-principal author with Galli and Greta Galli is daughter of Aurelio Galli.

This work was funded by grants MH070039, GM080403, HL122010, DA35263, DA38058 and NS007491-14 from the National Institutes of Health.