CCG•CGG interruptions in high‐penetrance SCA8 families increase RAN translation and protein toxicity

Abstract Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG•CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8, we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (n = 77). We show that repeat expansion mutations from individuals with multiple affected family members have CCG•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p‐eIF2α and polyAla and polySer RAN protein levels. Additionally, CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG•CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity, and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.

Thank you for the re-submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from the three reviewers who agreed to evaluate your manuscript. As you will see from the reports below, while the referee #1 is overall supporting publication of the manuscript, referees #2 and #3 acknowledge the interest of the study but also raise serious concerns that should be addressed in a major revision. Particular attention should be given to providing more insight in the underlying mechanism of the association of repeat interruptions with increased penetrance and age at onset.
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Yours sincerely, Zeljko Durdevic ***** Reviewer's comment s ***** Referee #1 (Comment s on Novelt y/Model Syst em for Aut hor): The work overall is of high technical qualit y and is of impact medically. Model syst em used in cells in cult ure which is fine. This is a fascinating study describing one potential mechanism that underlies complexities of SCA8. In this disease, there is a high lack of penetrance of repeat expansions. The authors analyze a large number of families and pedigrees and discover that there is no relationship between age of onset and repeat length, which is quite different from most other CAG-associated diseases. In analyzing the CAG-repeat sequences, they notice from a few families (one large family in particular), that there appears to be an association of CCG repeat interruptions with penetrance. Launching from this, they use various in vitro approaches to investigate potential molecular mechanisms. By comparing proteins that are pure polyQ or with an interrupted amino acid (Arg or gly) they document that the proteins have slightly higher toxicity bearing an interrupted amino acid. They compare RAN translation of constructs of pure vs interrupted repeats for proteins produced and see that more protein is produced from constructs with an interruption vs pure repeat sequence. They also examine folding and stability of the predicted RNAs with m-fold. Their discussion is particularly thoughtful. This is overall an interesting and well done study. Comments are largely minor, a few for additional rigor and most for clarification.
In the abstract, line 11, it would be helpful to indicate the experiments are in vitro. "At the molecular level, experiments in cells show that CCG etc.." because as a reader I was expecting that they were going to show this in human patient tissue.
In figure 1G, why the A vs AS individuals are listed in the order that they are is unclear (maybe there is a good reason). It seems clustering or grouping the A and AS individuals for each family would make the trend more clear and also make the point that this is one potential mechanism and there are likely others (which they do mention in the intro and discussion). Is it possible to asterisk in G the sequences corresponding to pg8, lines 11-20?
In Figure 3D, it is possible to perform dotblots with 1C2 as they did in figure 4A, to also assess protein level, in the advent that the protein is aggregated. Figure 3G should be quantitated, so it is clear how representative the images shown are. Same comment for Figure 4C.
In figure 5C, the legend is unclear. How do the patient alleles listed in the figure legend relate to the graph? And how does Figure 1G relate to that figure panel? Figure 1G is referred to in the legend. Table 1, Pg. 34, legend. They note that additional families were sequenced "and found to carry different interruptions." I presume they mean were not found to carry CGG interruptions or not only CGG interruptions? It is a bit unclear.
In figure EV1A, the open circles are not specified. Presumably those are individuals with pure CAG repeats that have disease.
In general, I suggest a number of the data in the supplemental data be moved into the main text. They are either very nice informative figures, or essential data that the reader definitely wants to see. These include EV1, EV2, EV3A, and Table EV1.
Referee #2 (Remarks for Author): This is a manuscript reporting that CGG interruption in SCA8 alleles explains penetrance: in highly penetrant families CGG repeat tends to present in CTG/CAG repeat expansion alleles. The authors' findings shed light on a long-term mystery why SCA8 repeat expansion shows low penetrance.
This review has some reservations. 1) Please clarify if longer CGG repeat caused stringer cell death shown in Figure 2. In LDH assay, pure Glutamine repeat caused cell death, while the cell death only slightly increased by CGG interruption. Also, how do the authors reconcile strong cell death with glutamine with low penetrance? 2) Please clarify if there were any other interruptions with different sequences (for example, TGG). 3) In Figure 3D, mobility of poly-glutamine and interrupted poly-glutamine is quite different while the length of expansions are the same. Is this what the authors expected? 4) This reviewer is wondering if the cell death induced by CGG interruption really explains penetrance. Ideally, it should be explained in gonadal cells. Does the CGG interruption make the repeat more unstable? 5) This reviewer suspects many readers would want to know how to diagnose SCA8 in respect of the repeat length, CGG interruptions and penetrance. Particularly, this work did not seem to clarify how we should consider about sporadic cases with SCA8 (ATXN8OS) CTG expansions. It may be better to show a table that classify how possible the diagnosis is with different numbers of CGG interruptions, number of affected individuals in his/her family.
Referee #3 (Comments on Novelty/Model System for Author): Use of an animal model system in addition to cell culture would greatly improve the significance of the findings.

Referee #3 (Remarks for Author):
This manuscript investigates the cause for incomplete penetrance in SCA8, a spino-cerebellar ataxia caused by a CTG•CAG expansion in the corresponding gene. By analyzing a large cohort of families in which the disease manifests with various penetrance, the authors convincingly show that repeat interruptions, in particular interruption by CCG•CGG, positively correlate with penetrance and age at onset. In the effort to elucidate the underlying mechanism, they provide some data showing that the presence of these interruptions: 1) increase RNA hairpin stability; 2) increase RNA poly-Ala and Poly-Ser; 3) increase the toxicity of the expressed protein, carrying poly-glutamine repeats. This part appears however still rather preliminary and the experiments performed fail to convincingly reveal the underlying mechanism.
Specific comments: 1) The experiments performed to tackle the reason why repeat interruptions are associated with increased penetrance and age at onset appear at this stage still preliminary. The authors express in cells constructs with similar length of pure or interrupted repeats and use as read-out LDH and MTT assays to assess cell death and viability. Since these experiments are all performed in overexpression, it is important to exclude difference in RNA levels, which is shown by the authors in the supplementary figures. However, in these experiments it is not clear what the individual data points in the figures 2 and 3 are. Are these independent transfections? Why there are many more experiments performed to assess viability and cell death than RNA level? Can this be the cause why some differences are significant (at p < 0.05) and others not? 2) Figure 3G and 4G use Hek293 cells to overexpress pure or interrupted poly-Gly or RAN poly-Ser. The authors emphasize that the expression pattern is different and this may the reason of increased toxicity, however there is no in-depth characterization of the different structures observed. What are the dotty structures in Figure 3G? What about the cytoplasmic staining that can also be observed? Is this pattern only seen in Hek293? Unfortunately, one does not learn anything about the reason for increased toxicity from these experiments.
3) The authors propose that the interruptions increase RAN translation. They show western blots or dot-blots (not clear why they use dot blots in one case) to prove this. It would be really important to show also how soluble these repeats are. The proteins in the insoluble pellets should be also shown and quantified.
We thank the reviewers for their careful comments and suggestions. We have now added considerable additional data to our manuscript and address these changes and each of the reviewer concerns below. We believe these additional data substantially improve our manuscript, which we hope is now suitable for publication in EMBO Molecular Medicine.

Referee #1 (Comments on Novelty/Model System for Author):
The work overall is of high technical quality and is of impact medically. Model system used in cells in culture which is fine.

Referee #1 (Remarks for Author):
This is a fascinating study describing one potential mechanism that underlies complexities of SCA8. In this disease, there is a high lack of penetrance of repeat expansions. The authors analyze a large number of families and pedigrees and discover that there is no relationship between age of onset and repeat length, which is quite different from most other CAGassociated diseases. In analyzing the CAG-repeat sequences, they notice from a few families (one large family in particular), that there appears to be an association of CCG repeat interruptions with penetrance. Launching from this, they use various in vitro approaches to investigate potential molecular mechanisms. By comparing proteins that are pure polyQ or with an interrupted amino acid (Arg or gly) they document that the proteins have slightly higher toxicity bearing an interrupted amino acid. They compare RAN translation of constructs of pure vs interrupted repeats for proteins produced and see that more protein is produced from constructs with an interruption vs pure repeat sequence. They also examine folding and stability of the predicted RNAs with m-fold. Their discussion is particularly thoughtful. This is overall an interesting and well done study. Comments are largely minor, a few for additional rigor and most for clarification.
We thank the reviewer for their positive comments and helpful suggestions.
1) In the abstract, line 11, it would be helpful to indicate the experiments are in vitro. "At the molecular level, experiments in cells show that CCG etc.." because as a reader I was expecting that they were going to show this in human patient tissue. We now state in the abstract that experiments were performed in cell culture: Page 2, lines 10-12: "At the molecular level, CCG•CGG interruptions increase RNA hairpin stability and in cell culture experiments increase p-eIF2a and polyAla and polySer RAN protein levels." 2) In figure 1G, why the A vs AS individuals are listed in the order that they are is unclear (maybe there is a good reason). It seems clustering or grouping the A and AS individuals for each family would make the trend more clear and also make the point that this is one potential mechanism and there are likely others (which they do mention in the intro and discussion). Is it possible to asterisk in G the sequences corresponding to pg8, lines 11-20?
We have now clustered the affected and asymptomatic individuals within each family in figure 1I (previously figure 1G) and have marked the sequence in the figure that corresponds to the allele described on page 7 line 13 (previously page 8 line 12) of the text with a "#" superscript.
3) In Figure 3D, it is possible to perform dotblots with 1C2 as they did in figure 4A, to also assess protein level, in the advent that the protein is aggregated.
We have now performed dot blots with the 1C2 antibody on the insoluble fraction and did not see polyGln proteins expression above the background (empty vector) levels for this antibody so we developed a second set of pure and interrupted CAG constructs where the TAG stop codons had been removed and a 3' HA epitope tag was added so that the polyGln protein could be detected using HA antibodies. We now include dot blots of insoluble 26th Jul 2021 1st Authors' Response to Reviewers polyGln protein detected by an HA-antibody and show that there are no significant changes in the levels of pure polyGln versus interrupted polyGln(Arg) proteins in the insoluble fraction (Fig EV2B-E): Page 11 lines 8-10: "Dot blots of insoluble protein fractions from HEK293T cells transfected with CAG-repeat constructs do not show significant differences in pure polyGln versus polyGln(Arg) levels ( Fig EV2B-E)." 4) Figure 3G should be quantitated, so it is clear how representative the images shown are. Same comment for Figure 4C.
We have now quantified the polySer aggregates and show that CGG interruptions increase the polySer aggregate burden per cell ( Figure 4E). We also quantify polySer protein levels by dot blot in cells transfected with the Pure 102 and Int. 104 constructs ( Figure 4A, C).  Fig 4D). Additionally, total aggregate burden is greater in cells expressing polySer(Arg) compared to pure polySer (p<0.01 for each experiment; Fig 4E)." We  Figure 1G relate to that figure panel? Figure 1G is referred to in the legend.
We have now added allele configurations to the X-axis labels in the legend for Figure 5C. We have also marked the sequences used in Figure 5C in Figure 1I with asterisks. Table 1, Pg. 34, legend. They note that additional families were sequenced "and found to carry different interruptions." I presume they mean were not found to carry CGG interruptions or not only CGG interruptions? It is a bit unclear.

6)
We have now updated the legend for Table 2 (previously Table 1) to specify the other types of interruptions that were seen in this study. We found one family with CGG interrupted alleles (Fig 1I Family 4)

7)
In figure EV1A, the open circles are not specified. Presumably those are individuals with pure CAG repeats that have disease.
We now state in legend to Figure 1B (previously Figure EV1A) that open circles indicate SCA8 patients with pure CAG repeat expansions.
8) In general, I suggest a number of the data in the supplemental data be moved into the main text. They are either very nice informative figures, or essential data that the reader definitely wants to see. These include EV1, EV2, EV3A, and Table EV1.
We have incorporated Figure Table EV1 to the main manuscript as Table 1. We really appreciate these suggestions and think that they improve the flow and readability of the manuscript.

Referee #2 (Remarks for Author):
This is a manuscript reporting that CGG interruption in SCA8 alleles explains penetrance: in highly penetrant families CGG repeat tends to present in CTG/CAG repeat expansion alleles. The authors' findings shed light on a long-term mystery why SCA8 repeat expansion shows low penetrance.
This review has some reservations. 1) Please clarify if longer CGG repeat caused stringer cell death shown in Figure 2. In LDH assay, pure Glutamine repeat caused cell death, while the cell death only slightly increased by CGG interruption. Also, how do the authors reconcile strong cell death with glutamine with low penetrance?
We thank the reviewer for these comments and separately answer each question below: 1a) Please clarify if longer CGG repeat caused stringer cell death shown in Figure 2 Because 2) Please clarify if there were any other interruptions with different sequences (for example, TGG).
We have now updated the legend for Table 2 (previously Table 1) to specify the other types of interruptions that were seen in this study. We found one family with CGG interrupted alleles (Fig 1I Family 4)  3) In Figure 3D, mobility of poly-glutamine and interrupted poly-glutamine is quite different while the length of expansions are the same. Is this what the authors expected? Based on previous observations, repetitive proteins (including polyglutamine proteins) often do not run at their expected molecular weights on SDS-PAGE gels. We find it interesting but not that surprising that the positively charged arginine interruptions change the conformation and mobility of the polar uncharged polyglutamine proteins.
To clarify this point we have modified the text on page 11, lines 3-5: "The change in mobility is likely caused by the introduction of positively charged arginine interruptions in these polar uncharged polyglutamine proteins."

4) This reviewer is wondering if the cell death induced by CGG interruption really explains
penetrance. Ideally, it should be explained in gonadal cells. Does the CGG interruption make the repeat more unstable?
We have now performed a transmission analysis which shows no significant difference in intergenerational repeat length changes for pure vs. CGG interrupted alleles for paternal or maternal transmission ( Figure EV1). Page 9, lines 3-6: "We saw no significant difference in changes in repeat length on paternal or maternal transmission of pure versus CCG•CGG interrupted alleles (paternal transmission p=0.5314, maternal transmission p=0.5748; Fig EV1)." 5) This reviewer suspects many readers would want to know how to diagnose SCA8 in respect of the repeat length, CGG interruptions and penetrance. Particularly, this work did not seem to clarify how we should consider about sporadic cases with SCA8 (ATXN8OS) CTG expansions. It may be better to show a table that classify how possible the diagnosis is with different numbers of CGG interruptions, number of affected individuals in his/her family. We thank the reviewer for this suggestion and have added Table 3 to clarify this point. We have also modified the discussion as follows: Page 16, lines 10-13: "For asymptomatic SCA8 expansion carriers, the risk of developing ataxia is increased by the presence of CCG•CGG interruptions, which more frequently occur in families with a prior history of ataxia. However, SCA8 ataxia patients may or may not have a family history of disease or sequence interruptions (Table 3)."

Referee #3 (Remarks for Author):
This manuscript investigates the cause for incomplete penetrance in SCA8, a spinocerebellar ataxia caused by a CTG•CAG expansion in the corresponding gene. By analyzing a large cohort of families in which the disease manifests with various penetrance, the authors convincingly show that repeat interruptions, in particular interruption by CCG•CGG, positively correlate with penetrance and age at onset. In the effort to elucidate the underlying mechanism, they provide some data showing that the presence of these interruptions: 1) increase RNA hairpin stability; 2) increase RNA poly-Ala and Poly-Ser; 3) increase the toxicity of the expressed protein, carrying poly-glutamine repeats. This part appears however still rather preliminary and the experiments performed fail to convincingly reveal the underlying mechanism.
Specific comments: 1) The experiments performed to tackle the reason why repeat interruptions are associated with increased penetrance and age at onset appear at this stage still preliminary. The authors express in cells constructs with similar length of pure or interrupted repeats and use as read-out LDH and MTT assays to assess cell death and viability.  (Fig 2D, E). These data demonstrate that the increased cell toxicity and the reduced cell viability that we see with the interrupted constructs are not caused by increased RNA levels.

Additionally, we now clarify in the methods section that MTT, LDH and RNA analyses were performed from a minimum of six independent transfections with each n representing a single independent transfection; for LDH and MTT assays, each independent transfection was performed in quintuplet.
Page 23, lines 5-7: "Toxicity and viability assays were performed in a minimum of six independent experiments and in each independent experiment the assays were performed in quintuplet." 2) Figure 3G and 4G use Hek293 cells to overexpress pure or interrupted poly-Gly or RAN poly-Ser. The authors emphasize that the expression pattern is different and this may the reason of increased toxicity, however there is no in-depth characterization of the different structures observed. What are the dotty structures in Figure 3G? What about the cytoplasmic staining that can also be observed? Is this pattern only seen in Hek293? Unfortunately, one does not learn anything about the reason for increased toxicity from these experiments.

First, we show that interrupted repeats increase the integrated stress response in transfected cells as described and discussed below.
Page 13, lines 7-9: "Additionally, we show in transiently transfected HEK293T cells that overexpression of interrupted repeats activated the integrated stress response (ISR) and increased p-eIF2a levels by 49% compared to pure repeats (p<0.05, Fig  4I, J)."  RAN proteins (Fig 4D). Additionally, total aggregate burden is greater in cells expressing polySer(Arg) compared to pure polySer (p<0.01 for each experiment; Fig 4E)." Third, we have quantified the number of intranuclear inclusions ( Figure 3J) Fig 3I, J), which are only rarely (>1%) found in cells overexpressing pure polyGln proteins (Fig 3J). These inclusions, which are found in transfected HEK293T and HeLa cells, colocalize with the nucleolar marker nucleophosmin (Fig 3K, EV2F)." Fourth, we discuss these new polyGln(Arg) data and to put them into context with data from other repeat expansion disorders, we have added the following paragraph to the Discussion: 3) The authors propose that the interruptions increase RAN translation. They show western blots or dot-blots (not clear why they use dot blots in one case) to prove this. It would be really important to show also how soluble these repeats are. The proteins in the insoluble pellets should be also shown and quantified.
We have now included matched Western and dot blots of soluble and insoluble fractions for RAN polySer ( Figure EV3A) and RAN polyAla ( Figure EV3F) and have modified the text as follows: Page 12, lines 9-14: "In the polySer reading frame, the GGC interruptions produce a polySer protein with glycine interruptions, polySer(Gly). Both pure polySer and polySer(Gly) proteins are highly insoluble with no protein detected in the soluble fraction (Fig EV3A). Dot blot analyses of the insoluble protein fraction showed 93.8% higher levels of interrupted RAN polySer(Gly) compared to pure RAN polySer proteins (p<0.01; Fig 4A, B)." Page 13, line 2-4. "The increases in polyAla protein levels did not result in overt changes in cellular localization ( Fig EV3D) and were found in both soluble and insoluble protein fractions (Fig EV3F)." Thank you for the submission of your manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) In the main manuscript file, please do the following: -Correct/answer the track changes suggested by our data editors by working from the attached document.
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Referee #3 (Remarks for Aut hor): The aut hors have subst ant ially revised the manuscript and addressed raised concerns.

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SCA8 families were included in our study if the repeat length and gender of the proband were known. Sample sizes for cell based work were chosen based on prior experience. All cell culture experiments were performed with a minimum of 3 biological replicates.
graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

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Patients for whom repeat size could not be determined or DNA was not available were not included in the study.
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Manuscript Number: EMM-2021-14095-V2
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