A Question of Axons

“I didn’t actually plan to study Down syndrome,” Ye says. It is spring 2023, and his lab is about to publish its seventh paper related to this chromosomal disorder. “But when we saw those effects of DSCAM, I started to realize how much was still unknown about the molecular and cellular mechanisms underlying the condition.”

DSCAM — or Down syndrome cell adhesion molecule — is the cellular protein he and Kim were studying when they went out for that coffee in 2012. At the time, there was no evidence that the molecule was involved in any of the characteristics of Down syndrome (its name was derived simply from the fact that the DSCAM gene that encodes the protein lies on chromosome 21 and is found in trisomy 21).

There was evidence, however, that mutations to DSCAM affected one particular aspect of neuronal development, and that’s what drew Ye to study it.

“It really started as a fundamental discovery question," explains Ye, now a research professor at the LSI and professor of cell and developmental biology at the U-M Medical School. "If we mutate this gene, what happens in the various parts of neurons?” 

Every neuron has three main parts: the cell center (or soma), where the nucleus resides; dendrites, which branch out from the soma to gather signals from other neurons; and axons, which extend out to send signals to other neurons through connections called synapses.

Genetic mutations that affect dendrites do not necessarily have the same effect on axons, and vice versa. That phenomenon was what Ye set out to study when he opened his lab in late 2008.

A lack of DSCAM was known to affect dendrites in Drosophila, or common fruit flies, which the Ye lab uses as a model organism. Because of their well-mapped nervous system and genetic similarities to humans, they can reveal important insights about the molecular and cellular mechanisms at play in many human disorders.

“We were looking at a bunch of different mutants to see if there was anything different between dendrites and axons, because they are in fact so different,” Kim recalls. “We were just testing here and there. And then we over- expressed this DSCAM, and we suddenly observed something very striking in the axons.”

What the researchers saw were axons that could not stop growing. Axons typically go through a period of rapid expansion during early brain development. But in the DSCAM mutants they kept growing well past the developmental checkpoint when they should have stopped.

This was the finding that Ye and Kim were discussing when they went to get coffee. They came up with an idea to directly compare levels of DSCAM proteins in neurons with the amount of axonal growth. When Kim mapped the data, he found a complete correlation: the more DSCAM protein present, the more the axons grew.

“Even at the time, we were not actually sure how this could be related to Down syndrome,” Kim says. “But with an additional copy, such as is found in Down syndrome, there is the opportunity to express more of those gene products. And we had shown that changing the level of this product had a dramatic effect on neurons.”

Ye’s curiosity was piqued: DSCAM levels are known to be elevated in patients with Down syndrome and other neurological disorders (including autism spectrum disorder, fragile X syndrome and some types of epilepsy).

But could DSCAM be contributing to the disorders by causing axon overgrowth?

To answer that question, he needed to enlist a more complex organism.

Ye had been a Drosophila researcher from his time as a postdoctoral fellow through the establishment and growth of his own lab. But the DSCAM findings in flies gave Ye the motivation he needed to approach his question in a mammalian system and the necessary clues about where to start.

“Flies are an excellent model for discovering disease mechanisms, but a fly is not just a tiny human with two wings,” he says. “They give us a great starting point. But to translate those mechanisms into information that’s more applicable to humans, we need an intermediary step.”

In 2015, he began the arduous work of creating a mouse model that could shed light on how DSCAM drives axon overgrowth in mammals, and whether it could be involved in Down syndrome and other neurological conditions.

When they analyzed the neurons of mice that had three copies of DSCAM, they found the same axonal overgrowth they observed in flies. But they also saw something new that explained how the axons were impairing neuronal function.

In mice, the overgrowth was taking place specifically on inhibitory neurons in the cerebral cortex — neurons that suppress activity in other neurons.

“When these neurons have increased synapses with another neuron, they actually have a dampening or quieting effect,” Ye says. “And we found not only increased axon growth, but also increased synapses with other neurons in the part of the brain that controls things like cognition and behavior.”

The team discovered that restoring DSCAM to normal levels reversed this effect. In mice that had only two copies of the DSCAM gene, but three copies of the other genes that are equivalent to human chromosome 21 genes, axon growth was normal.

This extensive project demonstrates the power of model organisms to provide new insights into human disease, Ye says: A basic science question tested in flies led to the discovery of the underlying genetic cause behind one characteristic of Down syndrome.

“Without the clear evidence we already had from Drosophila, we wouldn’t have known to test DSCAM effects in mice, and we wouldn’t have known to look at the axons. The fly work is what pointed us in the right direction,” he says.

It’s a model the Ye lab plans to replicate with more Down syndrome-related genes.

When scientists know or even suspect which gene leads to a single-gene disease in humans, they can turn to animal models to identify the exact cellular mechanisms that the gene affects to uncover potential targets for treatment.

“But approaches that are useful for studying single- gene diseases are not applicable to Down syndrome,” Ye explains. “There are over 200 functional genes on chromosome 21, and there is a long list of medical conditions associated with the syndrome. Our major challenge is figuring out which genes on chromosome 21 cause which medical conditions.”

In 2018, while still pursing the DSCAM research in mice, Ye returned to flies to tackle this challenge. With an award from the LSI’s Klatskin-Sutker Discovery Fund, he is assembling a legion of genetically modified fruit flies to begin matching individual genes to specific characteristics of Down syndrome. This philanthropic fund was established by a gift from the Klatskin and Sutker families to encourage this type of creative, early- stage research that has potential to have a positive impact on human health.

Because fruit flies have a very short life span, multiple generations can be created within a matter of weeks. And their well-mapped genome allows researchers to study the roles of specific genes, which leads to a better understanding of how those genes function in humans.

Starting with 60 genes expressed in the nervous system (Ye’s area of expertise), the Ye lab has created 60 different genetic lines of fruit flies — each over-expressing one gene found on chromosome 21. He has already uncovered one gene that interacts with DSCAM to cause changes in Drosophila neurons.

Ye hopes this project will point to more potential genetic culprits driving the various symptoms associated with Down syndrome in humans.