Blocking a common brain gas reverses autism-like traits in mice

A newly discovered biological chain reaction explains how high levels of a common brain chemical can lead to cellular overdrive in autism spectrum disorder. By tracing how nitric oxide disables a protective protein to accelerate cell growth pathways, researchers have identified a specific target that might one day yield new therapies. The findings were recently published in the journal Molecular Psychiatry.

Autism spectrum disorder involves differences in brain development that affect social communication and routine behavior. The biology behind these changes involves many genes and environmental factors. Researchers have observed that a signaling pathway called mTOR often runs unusually fast in the brains of autistic individuals.

The mTOR pathway acts as a central control center for cell growth, protein production, and energy use. When it functions properly, it helps brain cells build the connections needed for learning and memory. Yet the exact steps connecting autism risk factors to this hyperactive growth pathway have remained a mystery.

A team of scientists suspected that nitric oxide might be a missing link. Nitric oxide is a simple gas that helps brain cells communicate and regulates blood flow. High amounts of nitric oxide are often found in the brains and blood of people with autism.

When nitric oxide levels rise too high, the gas can attach directly to various proteins and alter how they work. This chemical tagging process is called S-nitrosylation. The research team wanted to see if this specific chemical tagging was responsible for pushing the cell growth pathways into overdrive.

The investigation was led by Shashank Kumar Ojha, a doctoral student, and Haitham Amal, a professor of brain sciences. Both researchers are based at the Hebrew University of Jerusalem. They designed a series of tests to map out exactly how nitric oxide interacts with the proteins that control cell growth.

The research team started by examining two different types of laboratory mice. These mice were genetically modified to lack either the Shank3 or Cntnap2 genes. Both gene mutations are associated with autism in humans and cause the mice to display similar behavioral traits.

Using specialized chemical tracking tools, Ojha and his colleagues looked at the proteins inside the brains of these mice. They focused on a specific protein called TSC2. In a healthy cell, TSC2 acts like a brake pedal for the mTOR growth pathway.

The researchers discovered that the mutant mice had unusually high amounts of nitric oxide attached to their TSC2 proteins. This nitric oxide tag acted like a signal that marked the brake protein for the cellular recycling center. As a result, the cells destroyed their own TSC2 proteins.

Without the TSC2 brake pedal, the mTOR growth pathway accelerated out of control. This overdrive forced the brain cells to manufacture proteins at an abnormal rate. This altered protein production disrupted normal brain cell function in both excitatory and inhibitory neurons.

To confirm this chain of events, the scientists treated the genetically modified mice with a drug that stops the brain from making nitric oxide. The results showed a clear mechanical link. Blocking nitric oxide prevented the destruction of the TSC2 brake protein.

With the brake protein intact, the cell growth pathway slowed down to a normal pace. The brain cells stopped overproducing proteins. The treatment successfully restored a natural balance to the cellular environment.

Ojha and his team then conducted a reverse experiment using normal mice without any genetic mutations. They gave these healthy mice a chemical that artificially activated the mTOR growth pathway. These mice soon began showing behavioral traits linked to autism.

The researchers placed the mice in a three-chambered box to test their sociability. The healthy mice treated with the pathway activator lost interest in interacting with unfamiliar mice. They preferred to spend time alone in an empty chamber.

The scientists also tested the mice in an elevated maze to measure anxiety levels. The mice with the activated growth pathway avoided the open areas of the maze. This behavioral shift confirmed that an overactive growth pathway alone can cause social deficits and anxiety.

The researchers also wanted to prove that the specific nitric oxide attachment point on the TSC2 protein was the root of the issue. They used a genetic technique to alter the brake protein in a way that prevented nitric oxide from attaching to it. They then injected this modified protein into the prefrontal cortex of the mutant mice.

This tiny genetic edit successfully protected the brake protein from being destroyed by nitric oxide. Consequently, the cell growth pathway returned to normal. The mice also became more social and spent more time exploring the open arms of the elevated maze.

To expand their research beyond animal models, the scientists grew human nerve cells in the laboratory. They engineered these human cells to carry the Shank3 genetic mutation. Just like the mouse models, these human cells showed a loss of the TSC2 brake protein and an overactive growth pathway.

Treating these human nerve cells with the nitric oxide blocker produced a familiar result. The drug protected the brake protein and calmed the cellular overdrive. This confirmed that the nitric oxide mechanism operates similarly in human tissues.

Finally, the researchers looked for this same pattern in actual patients. They analyzed blood plasma samples from autistic children alongside samples from neurotypical children. Some of the autistic children had specific Shank3 genetic mutations, while others had autism with no known genetic cause.

The human blood tests mirrored the laboratory experiments perfectly. The samples from the autistic children contained much lower levels of the TSC2 brake protein. Their blood also showed clear signs of an overactive mTOR growth pathway.

While these experiments provide a clear map of a cellular malfunction, the researchers note some limitations. The human blood samples came from a relatively small group of participants. Future studies will need to involve much larger groups of people to see if this pattern holds true across different types of autism.

Additionally, nitric oxide interacts with many different proteins in the body, not just the TSC2 brake protein. The researchers acknowledge that other chemical pathways might also play a role in the biological development of autism. They plan to investigate these other potential connections in upcoming projects.

Still, the discovery that blocking nitric oxide can restore normal cell function offers a tangible target for drug development. Scientists can now focus on creating medications that protect the TSC2 protein or safely reduce nitric oxide levels in the brain. This could eventually lead to interventions for individuals with specific genetic mutations.

As Amal explained in a press statement about the research: “Autism is not one condition with one cause, and we don’t expect one pathway to explain every case. But by identifying a clearer chain of events, how nitric oxide-related changes can affect a key regulator like TSC2 and, in turn, mTOR, we hope to provide a more precise map for future research and, eventually, more targeted therapeutic ideas.”

The study, “Nitric Oxide-Mediated S-Nitrosylation of TSC2 Drives mTOR dysregulation across Shank3 and Cntnap2 Models of Autism Spectrum Disorder,” was authored by Shashank Kumar Ojha, Maryam Kartawy, Wajeha Hamoudi, Manish Kumar Tripathi, Adi Aran, and Haitham Amal.