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/6J
Availability: Unknown.

A (G₄C₂) hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. Bidirectional transcription of this expansion yields sense (G₄C₂) and antisense (G₂C₄) repeat RNAs, which can then be translated—via repeat-associated non-ATG (RAN) translation—into a series of dipeptide repeat proteins.

(G₄C₂)₁₄₉ mice, generated by injecting an adeno-associated viral (AAV) vector encoding 149 G₄C₂ repeats into the brains of neonatal pups, represent the first model to accumulate dipeptide repeat proteins derived from both sense and antisense transcripts. The mice exhibit motor and cognitive dysfunction, and pathological hallmarks of ALS/FTD, including neuron loss, gliosis, aggregation of phosphorylated TDP-43, nucleocytoplasmic transport defects, accumulation of stress-granule proteins. This model may be useful for preclinical testing of therapeutics targeting stress granules, as well as sense or antisense RNA transcripts or dipeptide repeat proteins.

A unique feature of this model is the development of intranuclear foci containing antisense repeat RNA. Both sense and antisense RNA foci were found scattered throughout the brains of mice as young as 3 months of age, although the latter occurred less frequently—at 3 months, approximately 20 percent to 30 percent of cells in the motor cortex and hippocampus contained sense foci, but only 5 percent to 10 percent contained antisense foci. Cerebellar Purkinje cells were most affected by this pathology, with more than 50 percent of cells containing sense foci and approximately 20 percent containing antisense foci. RNA repeat foci were also found in the ventral horn of the spinal cord.

In parallel with the development of RNA foci containing sense and antisense transcripts, (G₄C₂)₁₄₉ mice also develop sense and antisense dipeptide repeat protein pathology. Mice exhibited an age-dependent accumulation of cytoplasmic aggregates containing poly-glycine-alanine (GA), poly-glycine-arginine (GR), and poly-glycine-proline (GP)—dipeptide repeat proteins derived from sense transcripts—in cortex, hippocampus, cerebellum, and spinal cord. Cytoplasmic inclusions containing poly-proline-alanine (PA) and poly-proline-arginine (PR), produced from antisense G₂C₄ transcripts, were also seen in the cortex and occasionally in the hippocampus, although these inclusions occurred much less frequently than inclusions containing proteins derived from sense transcripts.

(G₄C₂)₁₄₉ mice exhibit several additional neuropathological features of C9ORF ALS/FTD. Phosphorylated TDP-43 accumulates in cytoplasmic inclusions in the cortex and hippocampus, beginning by 3 months and increasing with age. The stress granule-associated proteins G3BP stress granule assembly factor 1 (G3BP1), ataxin-2, and eukaryotic initiation-factor 3η (eIF3η) were also seen to accumulate in the cortex and hippocampus of these mice. Mislocalization of RanGAP1, a regulator of nucleocytoplasmic transport, suggests that nucleocytoplasmic transport defects occur in mice as young as 3 months.

Astrogliosis is also seen in the cortices of 3-month-old mice. Cortical neuron loss is seen at 6 months, but not at 3 months.

Motor and cognitive deficits, assessed with hanging wire and contextual fear conditioning tests, respectively, emerged between 3 and 6 months. Hyperactivity in the open field was already apparent by 3 months.

Modification Details

An adeno-associated viral (AAV) vector was used to deliver 149 repeats of the hexanucleotide GGGGCC motif, along with the 5' (119 bp) and 3' (100 bp) flanking regions of the C9ORF72 gene, driven by the β-actin promoter. Virus was injected into the lateral ventricles of wild-type pups on postnatal day 0.

Phenotype Characterization

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