Disease Relevance: Amyotrophic Lateral Sclerosis, Frontotemporal Dementia
Strain Name: N/A
Genetic Background: C57BL/6
A G4C2 hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat expansion encodes five dipeptide repeat proteins that accumulate in ALS/FTD: glycine-arginine (GR), proline-arginine (PR), glycine-alanine (GA), proline-alanine (PA), and glycine-proline (GA). CamKII;(GR)80 mice were created to study the effects of poly(GR). Mice express 80 GR repeats; expression is restricted primarily to excitatory neurons in the cortex, and the timing of expression can be controlled by the experimenter. Mice exhibit age-related social deficits, neuron loss, microgliosis, DNA damage, and mitochondrial abnormalities. These phenotypes can be prevented or reversed by reducing levels of poly(GR) in adult mice.
CamKII;(GR)80 mice are produced by crossing a responder line carrying a (GGXCGX)80 sequence downstream of a tetracycline operon–responsive element (TRE) to an activator line expressing a tetracycline-controlled transactivator (tTA) under control of the CaMKIIα promoter. Bi-transgenic progeny constitutively express poly(GR) until transgene expression is suppressed by administration of the tetracycline analog doxycycline (DOX).
The integration site of the CaMKIIα-tTA transgene has been mapped (Goodwin et al., 2019). The transgene inserted on chromosome 12, resulting in a 508 kb deletion that affects five mouse genes: Vipr2 (vasoactive intestinal peptide receptor 2), Wdr60 (WD repeat-containing protein 60), Esyt2 (extended synaptotagmin-like protein 2), Ncapg2 (non-SMC condensin II complex, subunit G2), and Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2).
Three founder lines were created, and the line with intermediate expression—line 16—was selected for further study. There is an age-dependent accumulation of poly(GR) protein in the brains of these mice, with levels increasing at least to 12 months of age. The level of poly(GR) in the frontal cortices of 3- to 7-month-old mice is only about 5 percent to 15 percent that of patients with C9ORF72-related ALS/FTD.
Poly(GR) is seen primarily in the somata and dendrites of frontal cortex neurons. In the majority of these neurons, poly(GR) is diffusely distributed throughout the cytosol, but there are also neurons that contain aggregated poly(GR) and nuclear poly(GR).
In the description that follows, neuropathological, electrophysiological and behavioral features of CamKII;(GR)80 mice are compared with CamKII-tTA control mice.
Neuron loss occurs in the frontal cortices of CamKII;(GR)80 mice, beginning between 3 and 6 months of age. Dying neurons, recognized by the presence of cleaved caspase 3, were occasionally observed, and most of these neurons contained aggregated poly(GR).
Mitochondrial damage was apparent in 3-month mice and became more pronounced as the animals aged. Interestingly, poly(GR) was also observed inside mitochondria in neurons, and was found to bind to the ATP5A1 protein, a subunit of mitochondrial respiratory chain complex V. The activities of mitochondrial complexes I and V were found to be decreased in the frontal cortices of 6-month CamKII;(GR)80 mice.
By 6 months, poly(GR)-containing neurons frequently showed DNA damage.
TDP-43 inclusions were not seen in mice studied up to 8 months of age. Nor was there obvious evidence of nucleocytoplasmic transport defects in mice at least up to 6 months.
Microgliosis was evident at 6 months, but not 3 months, while astrogliosis became apparent between 6 and 9 months.
There was no difference in the amplitude of miniature excitatory postsynaptic potentials (mEPSCs) recorded from layer V pyramidal neurons in slices from 4.5-month control (CamKII-tTA) and CamKII;(GR)80 mice. However, mEPSCs occurred less frequently in CamKII;(GR)80 neurons, indicating synapse loss or a presynaptic deficit.
Deficits in social behavior, assessed using a three-chamber social interaction test, emerged between 3 and 6 months. Increased anxiety in the elevated plus maze also became apparent during this time. Working memory, assessed using the T maze, remained intact at least through 9 months of age.
Reducing levels of poly(GR) in adult mice ameliorates phenotypes
As noted above, expression of the (GGXCGX)80 transgene in CamKII;(GR)80 mice can be suppressed by feeding animals DOX. Shutting off expression of poly(GR) beginning at 1 month of age prevented the emergence of social deficits and reduced measures of anxiety in mice studied at 6 months of age. Administration of DOX between 7 and 9 months slowed or arrested the progression of disease-related cellular phenotypes, including the age-dependent increases in microgliosis, astrogliosis, DNA damage, and dying neurons containing cleaved caspase-3.
When visualized, these models will distributed over a 18 month timeline demarcated at the following intervals: 1mo, 3mo, 6mo, 9mo, 12mo, 15mo, 18mo+.
- Motor Impairment
- Cytoplasmic Inclusions
- Body Weight
- Lower Motor Neuron Loss
- NMJ Abnormalities
- Muscle Atrophy
- Premature Death
Cortical Neuron Loss
Neuron loss in the cortex, beginning between 3 and 6 months of age.
Lower Motor Neuron Loss
No data, but note that the transgene is not expressed in these neurons.
No TDP-43 inclusions seen in mice studied up to 8 months of age.
Microgliosis and astrogliosis evident at 6 and 9 months, respectively.
No difference between CamKII;(GR)80 mice and CamKII-tTA control mice up to 9 months of age.
No difference between CamKII;(GR)80 mice and CamKII-tTA control mice up to 11 months of age.
Last Updated: 01 Jul 2019
- Goodwin LO, Splinter E, Davis TL, Urban R, He H, Braun RE, Chesler EJ, Kumar V, van Min M, Ndukum J, Philip VM, Reinholdt LG, Svenson K, White JK, Sasner M, Lutz C, Murray SA. Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res. 2019 Mar;29(3):494-505. Epub 2019 Jan 18 PubMed.