Species: Mouse
Modification: Transgenic
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.

Neuropathology

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.

Synaptic function/electrophysiology

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.

Behavior

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.

Phenotype Characterization

COMMENTS / QUESTIONS

1. • Ludo Van Den Bosch
     Neurobiology, KU Leuven & VIB
   • Posted: 22 May 2019
   • Paper: C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

   This paper nicely shows that expression of a poly(GR) in all the layers of the cortex under the control of a CamKII promotor can induce an ALS/FTD-related phenotype. A major advantage of this study is that the poly(GR) is expressed at a relatively low level and that phenotypes induced by huge overexpression of the transgene are avoided.

   Another very interesting characteristic of the system used in this study is that the expression of poly(GR) can be reduced by feeding the mice with doxycycline. Interestingly, behavioral and even the cellular phenotypes can be significantly reversed when the expression of poly-GR is lowered. This is fascinating and has far-reaching consequences. It strongly indicates that therapeutic strategies lowering the expression of toxic dipeptide-repeat proteins can reverse disease phenotypes, even after disease onset. This reversal of the phenotype can be considered as a very interesting new insight, in addition to the systematic characterization of the underlying disease mechanism, which is related to mitochondrial dysfunction.

   View all comments by Ludo Van Den Bosch
2. • Adrian Isaacs
     University College London
   • Posted: 22 May 2019
   • Paper: C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

   This paper describes an interesting new poly(GR)-expressing mouse model. We will need a range of different models to ultimately understand the role of each DPR and their contribution to C9ORF72-repeat pathology, so this is a welcome addition.

   The most striking finding is that neuronal loss and synaptic dysfunction are identified even though GR is not detectable by immunostaining until six to eight months of age (although it can be detected earlier with ELISA). This suggests relatively low levels of likely soluble GR are sufficient to induce neurodegeneration.

   The paper also links poly(GR) to an early defect in mitochondrial function, which could explain the later development of DNA damage. Many pathways have been implicated in C9ORF72-repeat-induced neurodegeneration, and mitochondrial dysfunction should now be added as a new avenue for further investigation. Consistent with the previously reported AAV poly(GR) mouse model, no TDP-43 pathology was observed, and so the link between DPRs and TDP-43 is still an outstanding question.

   View all comments by Adrian Isaacs
3. • Robert Baloh
     Director of Neuromuscular Medicine, Cedars-Sinai Medical Center
   • Posted: 22 May 2019
   • Paper: C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

   This is a very nice addition to the building literature on mechanisms of toxicity from DPR expression in animal models. It brings to light a surprisingly specific mechanism, whereby poly(GR) alters mitochondrial ATP5A1, further linking mitochondrial dynamics and dysfunction to neurodegeneration and providing a potential therapeutic avenue.

   Challenges going forward for the field include interpreting this mechanism of toxicity in the context of the numerous other toxicities of the different DPRs, and determining whether some are more important than others, and where and when they are happening in human disease. 

   Regardless, it provides further support for the idea that diminishing gain-of-function products of the C9ORF72 repeat could have a therapeutic effect, and many of these strategies are in preclinical or clinical phase testing.

   View all comments by Robert Baloh
4. • Laura Ranum
     University of Florida
   • Posted: 22 May 2019
   • Paper: C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

   This work provides an additional mouse model and research tool for the C9 ALS/FTD community. Because C9 ALS/FTD disease is a complex disease, with the expression of two mutant transcripts (sense and antisense) and six dipeptide repeat-containing proteins, which are produced by repeat-associated non-AUG (RAN) translation, this polyGR-expressing mouse model is a simplified model for studying the potential contribution of polyGR to disease pathology.

   The paper provides a compelling data set showing that moderate expression of polyGR causes mitochondrial dysfunction that is linked to reductions in Atp5a1. In the future, it will be interesting to test if strategies described to reverse GR toxicity improve phenotypes in mouse models that express the repeat expansion and produce the multiple types of RAN proteins found in patients. It will also be of interest to test if Atp5a1 levels are reduced in these G4C2 mouse models and also in C9 patient tissues.

   View all comments by Laura Ranum

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References

Paper Citations

1. 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.

Further Reading