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Jul 27

New C9orf72 mouse models capture different aspects of ALS

Two new mouse models of ALS caused by mutations in C9orf72 display a range of physical and molecular characteristics of the disease.

Two new mouse models of ALS caused by mutations in C9orf72 display a range of physical and molecular characteristics of the disease. These new models, created independently by Packard scientists  Laura Ranum from the University of Florida and Clotilde Lagier-Tourenne at Harvard Medical School with Don Cleveland from the University of California in San Diego, gives scientists newer and better ways to test drugs that might potentially treat ALS and understand the cascade of events that leads to motor neuron degeneration.

When researchers first linked the repeat expansion in C9orf72 to ALS in 2011, they had no idea what the gene did or how it might contribute to ALS. Scientists around the world, including many funded by Packard, dug deep into the question. They discovered that the normal process that turns genes into proteins somehow got stuck, creating a buildup of toxic RNA foci and of small proteins called dipeptide repeats (DPRs). Identifying the downstream negative effects of the C9orf72 repeat expansion on a molecular level was one thing, but to truly understand how this mutation led to motor neuron degeneration and test potential therapies, researchers would need an animal model.

Building a good animal model of C9orf72 was difficult because the repeat expansion was inherently unstable. Researchers would insert a human C9orf72 gene that contained hundreds of repeats, but several generations later, it might only contain 30- not nearly enough to cause disease that researchers could study. Ranum and Lagier-Tourenne are the most recent scientists to have found their way around the problem using a bacterial artificial chromosome (BAC) to create the C9orf72 transgene, which provided more stability than other methods.

Ranum’s work, published in Neuron, created four different lines of BAC mice: C9-500/32 (two transgene copies, one with ~500 and the other with 32 repeats), C9-500 (one copy with ~500 repeats), C9-36/29 (four transgene copies with repeat sizes of 36 and 29 detected), and C9-37 (one copy with 37 repeats). Unlike other C9orf72 mouse models, the transgene inserted by Ranum and colleagues also contained substantial pieces of DNA flanking the C9orf72 gene. This DNA was likely to contain potential genetic elements that regulated the expression of the transgene and play an important role in pathology.

Three of the four mouse lines developed a buildup of both sense and anti-sense RNA (that is, RNA that’s transcribed in the normal and reverse direction, respectively) and DPRs. Up until four months of age, all of the C9orf72 mice appeared normal and healthy. At 16 weeks, however, the two expansion mouse lines showed gait abnormalities. Between 20 and 40 weeks, around one-third of female mice that carried the transgene with 500 repeats developed a rapidly progressing motor neuron disease with many of the same symptoms of ALS, including inactivity, labored breathing, sudden weight loss, hindlimb paralysis, and decreased survival.

A large proportion of the remaining mice developed a less severe, slower progressing form of disease. These mice showed denervation at the neuromuscular junction and a significant loss of motor neurons in the ventral horn of the lumbar spinal cord. Ranum and colleagues also observed significant behavioral changes, such as increased anxiety behaviors. The mice also displayed significant degeneration in the cerebellum and hippocampus.

The mouse model, then, captures both the molecular changes seen in ALS patients carrying the C9orf72 repeat expansion, as well as the physical and behavioral changes they experience, which will prove beneficial when testing potential ALS therapies.

“An effective therapy, one that is really working, you would expect would address the changes that we see in our mice. We’re looking forward to using it as a tool to identify drugs that are effective at preventing or reversing the neurodegenerative features that we see in our mice,” Ranum concluded.

The C9orf72 mouse model created by Lagier-Tourenne and colleagues, also published in Neuron, initially generated multiple mouse lines carrying a C9orf72 gene containing between 110 and 790 repeats. They narrowed that down to four different mouse lines: three that had 450 repeats but with different activations of C9orf72, and one line with only 110 repeats. Those mice that had more C9orf72 repeats had more buildup of both sense and anti-sense RNA in the nucleus than the mice with only 110 repeats. These mice also showed a buildup of dipeptide repeat proteins in neural tissue; again, the mice with 450 repeats had increased DPRs compared to the mice with 110.

Importantly, since the C9orf72 repeat expansion has been linked to both ALS and frontotemporal dementia (FTD), Lagier-Tourenne and colleagues tested the cognitive and behavioral effects of the C9orf72 repeat expansion in their mice. They found that the mice showed deficits in both spatial learning and working memory as they aged. Treating these mice with a small molecule called an antisense oligonucleotide (ASO), which specifically binds to and degrades the RNAs containing the C9orf72 repeats, decreased both the sense RNA foci and the number of DPRs.

A second set of experiments used mice carrying normal versions of C9orf72. When the researchers silenced one copy of the C9orf72 gene, the mice remained perfectly healthy. When they switched off both copies, however, the mice showed signs of immune dysfunction, confirming earlier work done by other Packard researchers. Although the mice weren’t perfectly healthy, they also weren’t showing signs of motor neuron disease, further emphasizing that there was something about the presence of the repeat expansion itself that was causing harm.

“This work is really helping us close in on how C9orf72 causes disease, and it’s also showing us that some of the molecular and behavioral signs associated with C9orf72 expansion can be modified with the use of ASOs,” Lagier-Tourenne said.