A newly discovered biological chain reaction explains how high levels of common brain chemicals cause cells to go into overdrive in autism spectrum disorders. By tracking how nitric oxide disables protective proteins and accelerates cell growth pathways, researchers have identified specific targets that may one day lead to new treatments. The results of this study were recently published in the journal molecular psychiatry.
Autism spectrum disorders involve differences in brain development that affect social communication and daily behavior. The biology behind these changes involves many genetic and environmental factors. Researchers observed that a signaling pathway called mTOR often operates abnormally fast in the brains of people with autism.
The mTOR pathway serves as a central control center for cell proliferation, protein production, and energy use. When this works properly, it helps brain cells build the connections needed for learning and memory. However, the precise steps linking autism risk factors to this hyperactive developmental pathway remain enigmatic.
A team of scientists suspected that nitric oxide might be the missing link. Nitric oxide is a simple gas that helps brain cells communicate and regulates blood flow. Large amounts of nitric oxide are often found in the brain and blood of people with autism.
If nitric oxide levels rise too much, the gas can attach directly to various proteins and change their function. This chemical tagging process is called S-nitrosylation. The research team wanted to see if this particular chemical tagging was responsible for driving cell proliferation pathways into overdrive.
The study was led by doctoral student Shashank Kumar Ojha and brain science professor Haitham Amal. Both researchers are based at the Hebrew University of Jerusalem. They designed a series of tests to determine exactly how nitric oxide interacts with proteins that control cell growth.
The research team started by examining two different types of laboratory mice. These mice are genetically modified to lack either the Shank3 gene or the Cntnap2 gene. Both gene mutations are associated with autism in humans and cause mice to display similar behavioral traits.
Ojha and his colleagues used specialized chemical tracking tools to examine the proteins in the brains of these mice. They focused on a specific protein called TSC2. In healthy cells, TSC2 acts like a brake pedal on the mTOR proliferation pathway.
The researchers found that the TSC2 protein in the mutant mice had abnormally large amounts of nitric oxide bound to it. This nitric oxide tag acted like a signal that marks brake proteins on cellular recycling centers. As a result, the cells destroyed their own TSC2 protein.
Without the TSC2 brake pedal, the mTOR growth pathway accelerated uncontrollably. This overdrive caused brain cells to manufacture proteins at an abnormal rate. This change in protein production disrupted normal brain cell function in both excitatory and inhibitory neurons.
To confirm this sequence of events, scientists gave genetically modified mice drugs that stopped the brain from producing nitric oxide. The results showed a clear mechanistic link. Blocking nitric oxide prevented destruction of the TSC2 brake protein.
When the brake protein was intact, cell proliferation pathways slowed to a normal pace. Brain cells stopped overproducing proteins. This treatment successfully restores the natural balance of the cellular environment.
Ojha and his team then performed a reverse experiment using normal mice without the genetic mutation. They gave these healthy mice a chemical that artificially activated the mTOR growth pathway. These mice soon began exhibiting behavioral traits associated with autism.
The researchers placed mice in a three-chamber box and tested their sociability. Healthy mice given the pathway activator lost interest in interacting with strange mice. They preferred to spend time alone in an empty room.
The scientists also tested mice in an elevated maze to measure anxiety levels. Mice with activated growth pathways avoided the open areas of the maze. This behavioral change confirms that only an excessive growth pathway can cause social deficits and anxiety.
The researchers also hoped to prove that a specific nitric oxide binding point on the TSC2 protein was the root of the problem. They used genetic technology to modify the brake protein in a way that prevents nitric oxide from attaching to it. They then injected this modified protein into the prefrontal cortex of the mutant mice.
This small gene edit successfully protects the brake protein from destruction by nitric oxide. As a result, cell proliferation pathways returned to normal. The mice also became more sociable and spent more time exploring the open arms of the elevated maze.
To expand their research beyond animal models, scientists cultivated human nerve cells in the laboratory. They engineered these human cells to carry the Shank3 gene mutation. Just like the mouse model, these human cells exhibited loss of the TSC2 brake protein and an exaggerated proliferative pathway.
Treating these human nerve cells with nitric oxide blockers produced familiar results. The drug protected brake proteins and quelled cellular overdrive. This confirmed that the nitric oxide mechanism works similarly in human tissue.
Finally, the researchers looked for this same pattern in real patients. They analyzed plasma samples from children with autism and samples from typically developing children. Some children with autism had a specific Shank3 gene mutation, while others had autism for which the genetic cause was unknown.
Human blood tests perfectly mirrored laboratory experiments. Samples from children with autism had much lower levels of the TSC2 brake protein. Their blood also showed clear signs of an overactive mTOR proliferation pathway.
Although these experiments provide a clear map of cellular dysfunction, the researchers note several limitations. Human blood samples were collected from a relatively small group of participants. Future studies will need to involve larger groups of people to see if this pattern holds true for different types of autism.
Additionally, nitric oxide interacts with a variety of proteins in the body, not just the TSC2 brake protein. Researchers acknowledge that other chemical pathways may also be involved in the biological development of autism. They plan to investigate these other potential connections in future projects.
Still, the discovery that blocking nitric oxide can restore normal cell function provides a concrete target for drug development. Scientists can now focus on developing drugs that protect the TSC2 protein or safely reduce nitric oxide levels in the brain. This could ultimately lead to interventions for individuals with specific genetic mutations.
In a press statement about the study, Amal explained: “Autism is not one condition with one cause, and we do not expect one pathway to explain all cases. However, we believe that nitric oxide-related changes may be linked to TSC2, By better identifying the sequence of events that affect key regulators such as mTOR, we hope to provide a more precise map for future research and, ultimately, more targeted treatment ideas.”
The study, “Nitric oxide-mediated S-nitrosylation of TSC2 causes mTOR dysregulation across Shank3 and Cntnap2 models of autism spectrum disorders,” was authored by Shashank Kumar Ojha, Maryam Kartawy, Wajeha Hamoudi, Manish Kumar Tripathi, Adi Aran, and Haitham Amal.
