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Around the same time as the TDP-43 discovery, Gitler arrived at Penn and set up a system to rapidly screen the effects of thousands of genes on yeast growth. When he spliced human TDP-43 into yeast, sure enough, clumps formed and the cells died early. Likewise, Bonini added human TDP-43 to her fruit flies, and the insects weakened, unable to move and climb normally. Those findings proved that yeast and fruit flies could serve as models of the deleterious effects of TDP-43 aggregation, an important confirmation. Bonini and Gitler hoped they could use the combined input of yeast and flies to address genes important for TDP-43 toxicity.
Gitler amped up a high-throughput genetic screening system, testing in TDP-43 yeast thousands of genes to see which, if any, accelerated or slowed cell death. They soon got an intriguing hit—the yeast homologue of a gene previously implicated in several human neurodegenerative conditions, ataxin-2.
When Bonini added ataxin-2 to her TDP-43 flies, they became even sicker; the gene clearly enhanced the toxic effects of TDP-43. "That was a 'Whoa' moment," says Bonini. "So we just went after ataxin-2."
The pair enlisted the help of clinicians who treat ALS, testing samples from 900 patients and 900 controls for variations in ataxin-2. What they found was remarkable: Some 5 percent of patients carried an altered version, whereas just 1.4 percent of controls did, as they reported in Nature. "It's a risk factor for ALS," says Bonini.
The discovery also hints at a common mechanism of neuronal damage caused by interactions between ataxin-2 and TDP-43 proteins. Previously, a long form of ataxin-2 had been implicated in a nerve disease called spinocerebellar ataxia type 2 (SCA-2). That disease, like ALS, also features clumps of TDP-43 protein in certain neurons. The 5 percent of ALS patients with abnormal ataxin-2, in contrast, carry a medium-long version of the gene. "It may be that interactions between these proteins are underlying different presentations of neurodegenerative disease, but the same pathway is involved," says Bonini. "There might be a continuum of damage caused by different versions of ataxin-2."
In a relatively short time, the pair has inched closer to understanding the causes of ALS. And even if they never fully understand what triggers the disease—a fear that Bonini expressed—their yeast and fly systems can help identify potential treatments. Already, they have identified several genes that appear to slow the toxic effects of TDP-43 and ataxin-2. If confirmed, these protective genes could inspire drug development, Bonini says.
In the laboratory of Arthur Horwich, mice engineered to carry the human SOD1 gene linked to hereditary ALS serve as subjects for drug testing. To generate ALS-like symptoms quickly in the animals, Horwich revved up expression of the SOD1 protein. "In mice you might need 50 to 100 times the concentration of [SOD1]," he says. "Then you get roughly the same level of toxicity you see accreted in humans over many decades." He adds, "Of all the neurological diseases, ALS is really the most accurately recapitulated in mice. We're beginning to see what the toxic effects are at the level of the neuron. We're trying to connect the dots."
One connection has become clear: in all types of ALS, whether linked to SOD1 or TDP-43, clumps of protein aggregate where they shouldn't in motor neurons. In 2009, Horwich published two papers showing that worms or mice carrying mutant human SOD1 produce aggregates in neurons similar to what ALS patients produce. Intriguingly, SOD1 mice show that motor neurons can, for a time, successfully clear the aggregates. But eventually, the cells' ability to disperse the clusters wears down and the nerves become dysfunctional. Horwich hopes some of the wide range of drugs he's testing on mice and worms rev up the cells' ability to clear the clumps. "If we can do that, the downstream toxicity is prevented and the animals walk away free," he says.
Bellen at Baylor College of Medicine is also trying to connect the molecular dots in mice bred with ALS-causing SOD1 mutations. Over the past decade, he's focused on the junction between motor neurons and muscle cells, studying a protein that may one day serve as an early warning sign of ALS. The protein, called VAPB, directs proper development of the neuromuscular junction, and it appears to be depleted in ALS patients and SOD1 mice, several other research groups have found. A critical segment of this protein also circulates in the blood and functions as a hormone, which makes it an ideal candidate for an early detection blood test. Bellen is now supplying his ALS mice with extra VAPB to see if the protein slows or reverses symptoms—a small, early step toward developing the protein as a drug.
Though progress identifying the causes of ALS has been slow and piecemeal, researchers are inching closer to a unified theory of the molecular mechanisms of the disease. "The field is making progress," Bellen says. "Every year we make strides toward better understanding. But it's been a very tough nut to crack."