Research Models

C9ORF72(AAV)(G4C2)66

Synonyms: 66-repeat mice, Petrucelli’s AAV C9 model

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Species: Mouse
Genes: C9orf72
Mutations: Hexanucleotide repeat in C9ORF72
Modification: C9orf72: Virus
Disease Relevance: Amyotrophic Lateral Sclerosis, Frontotemporal Dementia
Strain Name: N/A
Genetic Background: C57BL/6
Availability: Viral construct available through Leonard Petrucelli

Summary

When it was reported in 2015, this model was heralded as the first mouse model of ALS/FTD due to the repeat expansion in C9ORF72. In this model, an adeno-associated viral (AAV) vector is used to express the repeats in the CNS of wild-type mice. The construct contains 66 copies of the hexanucleotide motif, GGGGCC. The mice develop several pathological features of ALS/FTD, including nuclear RNA foci, dipeptide aggregates, and cytoplasmic inclusions of phosphorylated TDP-43. Subtle behavioral changes were also noted. By six months of age, the mice demonstrated anxiety-like behavior, antisocial beahvior, and hyperactivity (Chew et al., 2015).

By six months of age, the mice develop a number of brain pathologies reminiscent of C9ALS/FTD. RNA foci develop in the nuclei of neurons throughout the brain. Approximately 50 percent of the cells in the cortex, motor cortex, hippocampus, and cerebellar Purkinje cells contained RNA foci. Foci were also observed, albeit to a lesser extent, in the ventral horn of the spinal cord and other areas of the brain.

In addition to neuronal RNA pathology, the mice develop globular inclusions of RAN-translated dipeptides in neurons. Specifically, dipeptide repeats translated from sense RNA were detected; anti-sense dipeptides were not detected. Dipeptides from all frames were observed, including polyGA, polyGP, and polyGR. The majority of dipeptide inclusions were ubiquitin-positive and observed in neurons, although astrocytes had some as well.

Neurons also developed inclusions of phosphorylated TDP-43. Inclusions occurred in about 8 percent of cells in the cortex and hippocampus. Inclusions were primarily nuclear, with occasional cytoplasmic inclusions. TDP-43 pathology was largely restricted to cells containing RNA foci.

Neurodegeneration and gliosis were observed by six months of age. Compared with mice expressing just two repeats, the 66-repeat mice had 17 percent fewer neurons in the cortex and 11 percent fewer Purkinje cells in the cerebellum. At this age the neurons of the hippocampus, thalamus, and spinal cord were not affected. The number of GFAP-positive reactive astrocytes increased in the cortex. Overall, there was a modest decrease in brain weight compared with 2-repeat mice.

Subtle behavioral changes described as reminiscent of behavioral variant FTD (bv-FTD) were evident at six months of age. The 66-repeat mice displayed anxiety-like behavior in the open-field test, hugging the walls of the enclosure. They traveled faster and farther overall, indicating hyperactivity. They also showed abnormalities in social behavior, preferring an empty chamber to interaction with peers.

A subtle motor deficit was also seen at six months of age. On the Rotarod, 66-repeat mice performed as well as 2-repeat mice on the first day of testing. However, their performance failed to improve on subsequent trials, suggesting impairments in coordination and/or motor learning.

Modification Details

An adeno-associated viral (AAV) vector was used to deliver 66 repeats of the hexanucleotide GGGGCC motif. The construct does not contain the full-length C9ORF72 sequence. Virus was injected directly into the ventricles of wild-type pups on postnatal day 0. The β-actin promoter drives widespread expression in the brain and spinal cord.

Availability

Construct available through Leonard Petrucelli with MTA.

Phenotype Characterization

When visualized, these models will distributed over a 18 month timeline demarcated at the following intervals: 1mo, 3mo, 6mo, 9mo, 12mo, 15mo, 18mo+.

Absent

  • Lower Motor Neuron Loss

No Data

  • NMJ Abnormalities
  • Muscle Atrophy
  • Premature Death

Cortical Neuron Loss

Compared with mice expressing 2-repeats, the 66-repeat mice had 17 percent fewer neurons in the cortex at 6 months of age and 11 percent fewer Purkinje cells in the cerebellum. At this age neurons in the hippocampus and thalamus were not affected.

Lower Motor Neuron Loss

At 6 months, neuronal loss in the spinal cord was not detected.

Cytoplasmic Inclusions

By 6 months, inclusions of C9RAN dipeptides were present in neurons of the cortex and hippocampus, and to a lesser extent in the cerebellum and spinal cord. Inclusions contained polyGA, polyGP, and polyGR dipeptides and were largely ubiquitin-positive.

Gliosis

Astrogliosis in the cortex by 6 months.

NMJ Abnormalities

No data.

Muscle Atrophy

No data.

Motor Impairment

At 6 months, 66-repeat mice perform as well as 2-repeat mice on the Rotarod on the first day of testing. However, they fail to improve during subsequent trials, suggesting impairments in coordination and/or motor learning.

Body Weight

At 6 months females had a lower body weight than mice expressing 2-repeats. Body weight did not differ in males.

Premature Death

No data.

Last Updated: 11 Dec 2018

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References

Paper Citations

  1. Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME, Bieniek KF, Bauer PO, Whitelaw EC, Rousseau L, Stankowski JN, Stetler C, Daughrity LM, Perkerson EA, Desaro P, Johnston A, Overstreet K, Edbauer D, Rademakers R, Boylan KB, Dickson DW, Fryer JD, Petrucelli L. Neurodegeneration. C9ORF72 repeat expansions in mice cause TDP-43 pathology, neuronal loss, and behavioral deficits. Science. 2015 Jun 5;348(6239):1151-4. Epub 2015 May 14 PubMed.

Other Citations

  1. Leonard Petrucelli

Further Reading