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Apr 27
2016

New Mouse Models Yield Insight on C9orf72 Mutation

Two new mouse models, created with assistance from Packard researchers, provide new information on how the C9orf72 repeat expansion causes ALS

Since the discovery of the C9orf72 repeat expansion more than four years ago, one of the main goals of ALS research has been to develop an animal model to better understand what this gene does and how this mutation leads to ALS and frontotemporal dementia (FTD). A major question that remains to be resolved is whether the C9orf72 repeat expansion causes disease through the loss of its (as yet unknown) normal function, whether RNA and proteins generated by the expansion are themselves toxic, or both. The hundreds or thousands of repeated nucleotides in the repeat expansion are inherently unstable, which makes it hard for researchers to breed generations of mice with the same number of repeats.

In a pair of new studies in the journal Neuron, scientists from Cedars-Sinai Medical Center in Los Angeles and the University of Massachusetts Medical School (UMMS), and including Packard scientists Leonard Petrucelli of the Mayo Clinic and Fen-Biao Gao of UMMS, have created and characterized two different mouse C9orf72 models. The researchers then detailed the molecular, physiological, and behavioral characteristics of the mouse model, which contains anywhere from 100 to 1000 copies of the six-nucleotide C9orf72 gene repeat found in ALS/FTD. Although the molecular features of the model, such as RNA foci, directly match what is seen in many C9orf72 ALS and FTD patients, the mice did not develop behavioral signs of either disease. Nonetheless, the mice provide an opportunity to study ALS at the earliest stages of disease as well as test potential therapeutics for efficacy before moving into human trials.

In the first Neuron study, after first confirming that the mice contained between 100 and 1000 copies of the repeat expansion between exons 1a and 1b, the researchers at Cedars-Sinai Medical Center in Los Angeles then checked to see whether the mice showed some of the molecular features of the C9orf72 expansion seen in ALS patients. Because the expansion is so long and repetitive it confuses the cell’s transcription machinery, causing it to operate in the opposite direction, creating both sense and anti-sense RNA. This creates large numbers of RNA molecules as well as proteins called dipeptide repeats, in which two amino acids are repeated over and over. The transgenic C9orf72 mice carrying the repeat expansion showed both sense and anti-sense RNA foci in many neurons by three months of age, whereas the transgenic mice carrying a normal copy of the human C9orf72 gene did not. The expansion mice also showed a buildup of the dipeptide repeat proteins by 20 months of age, indicating that the proteins accumulated over time. When the researchers designed small molecules that would decrease C9orf72 RNA level, they were able to decrease both the number of RNA foci and dipeptide repeat proteins.

The expansion mice also showed some of the more subtle molecular signatures of ALS. These mice had an increased amount of a protein called nucleolin, involved in the synthesis and development of ribosomes, outside of its normal location in the nucleolus. Despite showing numerous of the molecular pathologies that accompany ALS patients, the expansion mice failed to show any differences in body weight, grip strength, or other physical and behavioral measures at a young or advanced age. The authors write in their study that the lack of physical signs of disease “is consistent with animal models of many neurodegenerative diseases, which typically mimic only certain aspects of the human pathophysiology and frequently lack overt neurodegeneration.”

In the second Neuron study, the scientists used a similar method to create two groups of transgenic mice, one that carried a normal copy of human C9orf72 and one that carried the gene with either 300 or 500 copies of the six nucleotide repeat expansion. As in the other model, these mice didn’t show any overt physical or behavioral signs of neurodegeneration, such as loss of weight or strength and early death. They also didn’t show any reactive changes to microglia or astrocytes, nor did the mice have accumulations of TDP-43 proteins in the cytosol or show increased sensitivity to cellular stress. Also similar to the first mouse model, the mice with the C9orf72 repeat expansion had sense and anti-sense RNA foci, along with clumps of dipeptide repeat proteins. Amounts of these foci and repeat proteins were also decreased when the researchers blocked production of the C9orf72 RNA with a virus expressing a small RNA that targeted a section of the gene.

The development of two new mouse models of C9orf72 ALS will not only provide a means to identify some of the specific pathological defects that accompany the earliest stages of ALS, often before patients are diagnosed, it will also give researchers new opportunities to test potential therapeutics before clinical trials, saving both time and money.