A novel, ataxic mouse model of ataxia telangiectasia caused by a clinically relevant nonsense mutation
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Abstract
Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated ( ATM ) and Aprataxin ( APTX ), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal’s first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm , we demonstrate that S mall M olecule R ead- T hrough (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.
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This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 4 2021, follows.
Summary
In this manuscript, the authors have generated a new mouse model for the severe disease, Ataxia Telangiectasia (A-T). They introduce null mutations in Atm onto the background of mice that are somewhat sensitized since they also harbor mutations in the Aptx gene. The outcome is the mice show a set of phenotypes that are strikingly similar to symptoms seen in human patients. These include cerebellar degeneration, cancer, and immune system abnormalities. The also deliver small molecule readthrough (SMRT) compounds into tissue explants and show that such a manipulation can restore the production of ATM protein. The success in producing an Atm model with cerebellar degeneration is a compelling advance as this …
This manuscript is in revision at eLife
The decision letter after peer review, sent to the authors on January 4 2021, follows.
Summary
In this manuscript, the authors have generated a new mouse model for the severe disease, Ataxia Telangiectasia (A-T). They introduce null mutations in Atm onto the background of mice that are somewhat sensitized since they also harbor mutations in the Aptx gene. The outcome is the mice show a set of phenotypes that are strikingly similar to symptoms seen in human patients. These include cerebellar degeneration, cancer, and immune system abnormalities. The also deliver small molecule readthrough (SMRT) compounds into tissue explants and show that such a manipulation can restore the production of ATM protein. The success in producing an Atm model with cerebellar degeneration is a compelling advance as this particular phenotype has been incredibly difficult to reproduce in animal models. The authors perform an interesting set of analyses to confirm that the other important features of the disease are also present in their mice. This paper has broad interest to multiple fields including neuroscience, cancer, and immunology.
Essential Revisions
It is not clear how progressive the cerebellar degeneration is. What is the spatiotemporal pattern of degeneration? Please consider the lobule by lobule effects over time.
For the electrophysiology, what stage cells have you recorded from? That is, what was the structure of the Purkinje cells that you recorded? If the cells look really "normal" but fire abnormally, then please comment on how they are being affected. If the morphology is abnormal, then please explain what defects you see and how they might impact function. Essentially, the authors need to disentangle cell autonomous effects and non-cell autonomous effects with more clarity. That is, are you studying the "dying" cells or the cells that that escaped the genetic defect?
Are both the Atm and the Aptx genes expressed in all (or the same) Purkinje cells? What is the experimental evidence?
Please provide more context and rationale for Aptx in the abstract. As it stands, its mention comes out of nowhere.
In the Introduction, please provide more information as to why previous studies/models might have failed to produce severe Atm-related cerebellar phenotypes.
In the Introduction, the rationale for the choice of paring the Atm mutations with defects in the Aptx gene is unclear. Are they in the same pathway? Are the genes located in close proximity to one another? There are many issues that need to be discussed.
Related to above, ATM and APTX, while involved in DDR, are involved in parallel pathways-ATM in DNA double stranded break repair, and APTX in single stranded break repair. Homozygous mutations in APTX causes human ataxia (AOA1), but there is nothing to indicate an intersection mechanistically between AT and AOA1. One could just as well call the AT-APTX double mutation a model of AOA1. As indicated above, please expand on the rationale of the experimental design.
Also, are there more single stranded DNA breaks? Double stranded DNA breaks? Is there a sequestration of SS DNA break repair components including PARP1? How are the changes in PC firing related to DDR (it would be worthwhile for the authors to examine the following papers Hoch et al. Nature. 2017 Jan 5;541(7635):87-91, Stoyas et al. Neuron 2020 Feb 19;105(4):630-644) to give insight into studies that can explore mechanism for DDR and changes in cerebellar morphology/function.
Therefore, the authors need to address whether single vs double stranded break repair is present and the authors could do a better job of linking the change in PC firing to DNA damage.
Figure 2B: Apologies if I am missing something, but I do not understand the reason or explanation for what determines the probability of survival for the green, gold, and orange traces (the three severe cases in the graph). That is, why is the gold so strong?
How come rotarod was not used as a test? This is a standard motor behavior test that is useful for comparing across animal models and studies.
Related to above, why not use in vivo recordings? I can understand using slice recordings to tackle the biophysical and intrinsic mechanisms, although the authors did not do that. It seems to me that extracellular recordings would have been more informative in the in vivo, awake context.
The authors picked specific regions of the cerebellum to target their slice recordings, which is perfectly reasonable. But why did you pick these regions? Please provide a full justification and discussion for the importance of these particular lobules in relation to what you are trying to solve.
Given the use of slice recordings and that Purkinje cell degeneration is a key aspect of the phenotype, it would be very compelling if the authors showed some filled cells. As it stands, it is very hard to appreciate what the severity of neuropathology actually looks like, especially in relation to what the functional defects are teaching us.
The authors state that "The largest differences were detected in the anterior [38.6{plus minus}3.4 Hz (n=187) vs. 88.1{plus minus}1.8 Hz (n=222)] and posterior [46.9{plus minus}1.9 Hz (n=175) vs. 84.1{plus minus}2.4 Hz (n=219)] medial cerebellum [1-way ANOVA, p<0.0001; Fig. 4B]." Okay, but what does this mean? What is your interpretation for why these regions were more heavily impacted (cell sensitivity based on circuit architecture, gene expression and protein make-up, neuronal lineage?) and how might it impact the phenotype?
The authors state and reference "Previous studies in mouse models of heritable ataxia indicate that physiological disruption in PN firing not only includes changes in frequency but also affects its regularity (Cook, Fields, and Watt 2020)." I agree with having this reference, but what about other models of ataxia? There are a number of other excellent models that should be discussed.
Purkinje cell firing data (figure 4B) should not be averaged across all of the ages, as this is not standard practice, and would be akin to averaging all behavior across ages. I think the data in fig. 4C suffices. If you want to compare across lobules on one graph, simply choose a particular age (perhaps when behavioral changes are first observed?) or at the oldest age.
Why examine Purkinje cell firing deficits in different lobules but not make that distinction for Purkinje cell loss? The Purkinje cell loss analysis focussed on the areas with most pronounced firing deficits but this means that we don't know whether the cells that fire abnormally are the only ones that die. Also see point #2 above.
Figure 4E and related text: Please provide a much more extensive set of images to show the cerebellar pathology. 1) Please show views of the different lobules to demonstrate the pattern of degeneration. 2) Please show different ages to show the progression of degeneration. 3) Please show higher power images of the Purkinje cells to clearly demonstrate their morphology.
The authors need to need provide more data for what is actually happening in relation to cell death. Why not perform Tunel or caspase staining etc.? The authors must show that there are actually acellular gaps where cells have died, or some other indication that cell death has occurred or is occurring.
Also in relation to the Purkinje cell degeneration, what do the dendrites look like? What about the axons? Do you see any torpedoes or axonal regression?
In regards to the cerebellar degeneration, what happens to the other cell types in the cerebellar cortex? Are they intact? What about the cerebellar nuclei?
The authors state "Of interest, APTX deficiency by itself had the greatest effect on the loss of DN4 cells...". Okay, but it is hard to see what this means for A-T as a disease. Interesting as it is, what is the relevance of this gene and these findings to the actual disease?
Please provide a more extensive description and rationale for why this explant system was chosen.
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