The Johns Hopkins team further reveals how a mutation of Shank3 leads to altered communication between brain cells at synapses — those gaps between cells across which electrical information flows.
The team first identified Shank proteins in studies of synaptic proteins that are regulated by neuronal activity. The scientists say they were motivated to make the new mouse after other investigators showed that a subset of children with autism carries mutations of the Shank3 gene. Patients with a mutation that prevented Shank3 proteins from forming complexes at the synapse had particularly severe disease despite having one mutant and one normal copy of Shank3. To refine a mouse model of the disease that mimicked the human mutation, the researchers altered one copy of Shank3 and left intact a normal copy of the gene.
“We hope our model of accelerated protein degradation will be applicable to other genetic causes of autism and, perhaps, schizophrenia” says Paul Worley, M.D., a professor of neuroscience at the Johns Hopkins University School of Medicine. “Behavioral changes in these mice can be understood to be the result of the changes in synaptic proteins.”
Ali Bangash, a graduate student of biological chemistry and molecular biology working in Worley’s lab, used biochemical methods to survey the levels and activity of the Shank3 protein in sections of brain from the Shank3 mutant mouse. He discovered that the amount of normal Shank3 protein was dramatically reduced in the new Shank3 mutant. In addition, the mutant Shank 3 protein seemed to cause any normal Shank3 protein to be targeted for rapid degradation. In fact, the amount of Shank3 that makes its way to where it is supposed to be (at synapses in the brain) is reduced by 90 percent compared to normal mice, according to Worley.
“By mutating one copy of Shank3, we didn’t simply lose half of the amount of protein that normally would be produced, we lost more of good protein,” Worley says, adding that the team also noted behavioral and electrophysiological changes when they mutated one copy of Shank3 in mice.
In one experiment, the researchers electrically stimulated nerve cells from the mouse brains, assessed the strength of synapses from Shank3 mutants and compared these to brain tissue from normal, wild-type mice. The scientists found that the ratio of two different types of proteins vital to memory and learning was out of whack in Shank3 mutants.
The team also assessed the behaviors of the Shank3 mutants by using social interaction tests and comparing them to wild-type mice. In one test, a Shank3 mutant was placed with another mouse in a cage, leaving both free to explore their surroundings. The Shank3 mutant mouse spent much of the time shying away from social interaction. The wild-type showed an increased level of interest, indicated by time spent sniffing the Shank3 mutant. However, when the Shank3 mutant was allowed to roam free while the other mouse was confined under a dome with slits, the Shank3 mouse showed increased interest, exploring the immobilized mouse more than a wild-type did under the same circumstances. The investigators say this behavior is consistent with characteristics of an autism-like disorder.
In another test, a younger mouse was introduced three times to a Shank3 mutant and to a wild-type mouse. The wild-type and Shank3 mouse both spent less time exploring the young mouse on each successive interaction, indicating they got used to each other. When a different “surprise” mouse of the same age and genetic background was brought in, the wild-type mouse re-explored the animal with renewed interest, apparently recognizing it as “new.” The Shank3 mutant, also apparently recognizing it as new, not only re-explored, but also became markedly aggressive, nipping, and biting the “surprise” mouse, a behavior that the investigators concluded was also autism-like.
Worley says it is “exceedingly challenging” to make an accurate animal model of autism, a complex neurobiological disorder that inhibits a person’s ability to communicate and develop social relationships.
Autism spectrum disorders are diagnosed in one in 110 children in the United States, and one in 70 boys. According to Autism Speaks, North America’s largest autism science and advocacy organization, the prevalence of autism has increased 600 percent in the past two decades. The Centers for Disease Control and Prevention have called autism a national public health crisis, the cause and cure of which remain unknown.
This work was supported by National Institute of Neurological Disorders and Stroke, National Institute of Mental Health, Autism Speaks, National 973 Basic Research Program of China, The Autism Science Foundation, The Hartwell Foundation, and the NIDCD Intramural Program.
In addition to Worley and Bangash, other Johns Hopkins authors are Joo Min Park, Tatiana Melnikova, Soo Kyeong Jeon, Deidre Lee, Sbaa Syeda, Juno Kim, Joshua Schwartz, Jian Cheng Tu, Jia-Hua Hu, David J. Linden, Alena Savonenko, and Bo Xiao.
Authors from Sichuan University, Chengdu, China, are Dehua Wang, Yiyuan Cui and Xia Zhao. Authors from the University of Texas Southwestern Medical Center are Mehreen Kouser, Haley E. Speed, Craig M. Powell and Sara E. Kee. And, from the National Institutes of Health, Ronald S. Petralia.
Material adapted from Johns Hopkins Medicine.