Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism

Abstract This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan‐reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image‐based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy.


2nd Jun 2021 1st Editorial Decision
Dear Dr. Kakhlon, Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from two of the three reviewers who agreed to evaluat e your manuscript . Given that referee #2 will unfort unat ely not be able to ret urn his/her report in a timely manner, and that bot h referees #1 and #3 are overall posit ive, we prefer to make a decision now in order to avoid furt her delay in the process. As you will see from the report s below, the referees acknowledge the int erest of the st udy but also raise import ant concerns that should be addressed in a major revision.
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Yours sincerely, Zeljko Durdevic Editor EMBO Molecular Medicine ***** Reviewer's comment s ***** Referee #1 (Comment s on Novelt y/Model Syst em for Aut hor): Adult Polyglucosan Body Disease (APBD) is a neurological disorder that affect s mult iple tissues. Current ly there is no curat ive treat ment for pat ient s wit h APBD. Kakhlon et al. report ed the effect iveness and possible mechanisms of act ion of the polyglucosan-reducing compound 144DG11 in APBD mice and APBD pat ient cells. This therapeut ic approach has the pot ent ial to cure pat ient s wit h APBD and ot her polyglucosan body diseases. The experiment s were well designed and conduct ed and the manuscript was well writ ten.
Referee #1 (Remarks for Aut hor): Adult Polyglucosan Body Disease (APBD) is a neurological disorder that affect s mult iple tissues. Current ly there is no curat ive treat ment for pat ient s wit h APBD. Kakhlon et al. report ed the effect iveness and possible mechanisms of act ion of the polyglucosan-reducing compound 144DG11 in APBD mice and APBD pat ient cells. This therapeut ic approach has the pot ent ial to cure pat ient s wit h APBD and ot her polyglucosan body diseases. The experiment s were well designed and conduct ed and the manuscript was well writ ten. Comment s: 1. Liver glycogen content varies greatly if the mice were not fasted. The authors need to indicate whether the mice were fasted prior to euthanization. 2. Figure 2 shows that 144DG11 had no apparent effect on muscle polyglucosan bodies. What muscle was examined? Did you look at different skeletal muscles as well as smooth muscle (e.g., bladder)? 3. In Figure 6E, the LAMP1 KO was probably a typo of LAMP1 KD. 4. Figure7D should be Figure 7E in the paragraph on page 13 "As shown in Figure 7D, under 48 h starvation 12.2% and 6.8% of the 2,898 proteins analyzed were respectively up and down modulated in APBD-patient as compared to HC cells. Interestingly, endocytosis, a pathway implicated in lysosomal biogenesis and function, is a KEGG pathway upmodulated in APBD cells ( Figure S9), while oxidative phosphorylation is down-modulated in APBD cells ( Figure S10). As an important control, GBE was indeed down-modulated in the APBD cells ( Figure 7D)". 5. Last paragraph on page 17 "Importantly, lysosomal glycogen degradation takes place in parallel with its cytoplasmic degradation (37), and, specifically, in a GSDIV mouse model, which also models APBD in mice, overexpression of the lysosomal glycogenase α-glucosidase corrected pathology (38)." This is inaccurate description. Consider to revise to "... overexpression of human GDE by gene therapy corrected pathology in skeletal muscle, liver, and brain (38) and treatment with recombinant human lysosomal enzyme acid α-glucosidase alleviated disease in the liver (Yi et al., Alglucosidase alfa treatment alleviates liver disease in a mouse model of glycogen storage disease type IV. Mol. Genet. Metab. Rep., 2016).

Referee #3 (Comments on Novelty/Model System for Author):
While the work carried out by this team concerns both in vivo and in vitro approaches which is a real added value, the murine model could be better exploited.
Referee #3 (Remarks for Author): The authors tested both in vivo and in vitro a glycogen lowering therapeutic molecule -144 DG11obtained by high throughput screening. In the first part, the molecule is tested in the Gbe ys / ys murine model. Motor performances were improved and a significant reduction in polyglucosan bodies were observed in the brain, liver, heart, and peripheral nerves except muscle. By using indirect calorimetry, they demonstrated a shift of the respiratory quotient towards carborhydrate catabolism. The putative mechanisms of action were then deciphered in skin fibroblasts from APBD patients. The combination of the two approaches in vivo and in vitro is undoubtedly an added value. The data obtained in vitro appear to be well controlled and robust. The murine model could however be much better exploited, in particular with regard to the results obtained in vitro on autophagy, LAMP 1 and the lysosome.
Major recommendations : In the mouse model of APBD (Gbe ys/ys), please investigate: -the lysosomal compartment both in liver or heart and skeletal muscle from Gbe and wt mice by using immunohistochemistry against LAMP1 and by quantification of LAMP1positive-areas for example -autophagy through LC3 and p62 for liver or heart and skeletal muscle by using immunohistochemistry against LC3 and by quantification of LC3 -vacuolated area positive or by Western-blot for example.
Please rephrase the in vivo results p5, P6 and p7. It is sometimes very confusing to understand at what age the mice were injected and at what age they were sacrificed, and the results obtained. A schematic representation of the experimental design would be appreciated as an additional figure.
***** Reviewer's comments ***** We thank the reviewers for their constructive criticisms. We have made all requested changes, which undoubtedly improved the quality of our manuscript. Please find below the respective comments our replies and references to the relevant manuscript modifications.

Referee #1 (Comments on Novelty/Model System for Author):
Adult Polyglucosan Body Disease (APBD) is a neurological disorder that affects multiple tissues. Currently there is no curative treatment for patients with APBD. Kakhlon et al. reported the effectiveness and possible mechanisms of action of the polyglucosan-reducing compound 144DG11 in APBD mice and APBD patient cells. This therapeutic approach has the potential to cure patients with APBD and other polyglucosan body diseases. The experiments were well designed and conducted and the manuscript was well written.

Referee #1 (Remarks for Author):
Adult Polyglucosan Body Disease (APBD) is a neurological disorder that affects multiple tissues. Currently there is no curative treatment for patients with APBD. Kakhlon et al. reported the effectiveness and possible mechanisms of action of the polyglucosan-reducing compound 144DG11 in APBD mice and APBD patient cells. This therapeutic approach has the potential to cure patients with APBD and other polyglucosan body diseases. The experiments were well designed and conducted and the manuscript was well written.

Comments:
1. Liver glycogen content varies greatly if the mice were not fasted. The authors need to indicate whether the mice were fasted prior to euthanization.
Thank you for this comment. We agree. Glycogen levels can change considerably depending on the feeding regime. The mice did not fast before their euthanasia. This is now mentioned under Methods, In vivo experiments, on p. 20.
2. Figure 2 shows that 144DG11 had no apparent effect on muscle polyglucosan bodies. What muscle was examined? Did you look at different skeletal muscles as well as smooth muscle (e.g., bladder)?
We investigated the quadriceps as a representative striated skeletal muscle. This skeletal muscle is usually investigated in APBD models as the disease is a neuromuscular disorder, which affects motor abilities. 3. In Figure 6E, the LAMP1 KO was probably a typo of LAMP1 KD.
Correct. Thanks for pointing this out. We corrected that. Figure 7E in the paragraph on page 13 "As shown in Figure 7D, under 48 h starvation 12.2% and 6.8% of the 2,898 proteins analyzed were respectively up and down modulated in APBD-patient as compared to HC cells. Interestingly, endocytosis, a pathway implicated in lysosomal biogenesis and function, is a KEGG pathway upmodulated in APBD cells ( Figure S9), while oxidative phosphorylation is down-modulated in APBD cells ( Figure S10). As an important control, GBE was indeed down-modulated in the APBD cells ( Figure 7D)".

Corrected.
3rd Aug 2021 1st Authors' Response to Reviewers 5. Last paragraph on page 17 "Importantly, lysosomal glycogen degradation takes place in parallel with its cytoplasmic degradation (37), and, specifically, in a GSDIV mouse model, which also models APBD in mice, overexpression of the lysosomal glycogenase α-glucosidase corrected pathology (38)." This is inaccurate description. Consider to revise to "... overexpression of human GDE by gene therapy corrected pathology in skeletal muscle, liver, and brain (38) and treatment with recombinant human lysosomal enzyme acid αglucosidase alleviated disease in the liver (Yi et al., Alglucosidase alfa treatment alleviates liver disease in a mouse model of glycogen storage disease type IV. Mol. Genet. Metab. Rep., 2016).
Corrected. We cited there the wrong reference by Yi et al. Thank you for bringing that to our attention.

Referee #3 (Comments on Novelty/Model System for Author):
While the work carried out by this team concerns both in vivo and in vitro approaches which is a real added value, the murine model could be better exploited.

Referee #3 (Remarks for Author):
The authors tested both in vivo and in vitro a glycogen lowering therapeutic molecule -144 DG11 -obtained by high throughput screening. In the first part, the molecule is tested in the Gbe ys / ys murine model. Motor performances were improved and a significant reduction in polyglucosan bodies were observed in the brain, liver, heart, and peripheral nerves except muscle. By using indirect calorimetry, they demonstrated a shift of the respiratory quotient towards carborhydrate catabolism. The putative mechanisms of action were then deciphered in skin fibroblasts from APBD patients. The combination of the two approaches in vivo and in vitro is undoubtedly an added value. The data obtained in vitro appear to be well controlled and robust. The murine model could however be much better exploited, in particular with regard to the results obtained in vitro on autophagy, LAMP 1 and the lysosome.

Major recommendations :
In the mouse model of APBD (Gbe ys/ys), please investigate: -the lysosomal compartment both in liver or heart and skeletal muscle from Gbe and wt mice by using immunohistochemistry against LAMP1 and by quantification of LAMP1positive-areas for example -autophagy through LC3 and p62 for liver or heart and skeletal muscle by using immunohistochemistry against LC3 and by quantification of LC3 -vacuolated area positive or by Western-blot for example. Figure 6 new panels (current Fig. 6C and 6G) analyzing LAMP1 and autophagy (LC3 and p62) in liver and skeletal muscle sections taken from 144DG11 and vehicle treated Gbe ys/ys mice. As opposed to skeletal muscle, 144DG11 was both biodistributed ( Figure  2C) and effective (Figures 2A, 2B, and 6B) in the liver of Gbe ys/ys mice. Therefore, we compared the effects of 144DG11 on our fixed and deparaffinized muscle and liver sections derived from 144DG11 and vehicle treated Gbe ys/ys mice. As expected, LC3 and p62 levels were indeed reduced by 144DG11 only in liver, and not in muscle, sections. In addition, LAMP1 staining area was reduced by 144DG11 only in liver sections, possibly associated with the compound-mediated improvement of lysosomal function.

We accept the reviewer comment and added in
Please rephrase the in vivo results p5, P6 and p7. It is sometimes very confusing to understand at what age the mice were injected and at what age they were sacrificed, and the results obtained. A schematic representation of the experimental design would be appreciated as an additional figure. Figure 1K.

9th Aug 2021 1st Revision -Editorial Decision
Dear Dr. Kakhlon, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendment s: 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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12th Aug 2021 2nd Authors' Response to Reviewers
The authors performed the requested editorial changes.

13th Aug 2021 2nd Revision -Editorial Decision
We are pleased to inform you that your manuscript is accept ed for publicat ion and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine. Do the data meet the assumptions of the tests (e.g., normal distribution)? Describe any methods used to assess it.
Is there an estimate of variation within each group of data?
As in 1a, minimal sample sizes in animal studies were also determined retrospectively based on the means and SD obtained. In most cases, at the means and SD obtained, a sample size of n=5 was already sufficient to provide a power of at least 80% (Charan and Kantharia). Our actual sample sizes were 7-9, so that statistical power was satisfactory.
Animals were excluded from the experiment if weight was reduced by >10% between sequential weightings or by >20% from initiation. These criteria were pre-estblished.
The experimenters who measured motor and histological parameters were blinded to the treatments, which were encoded by letters and numbers not revealing the treatment.

Manuscript Number: EMM-2021-14554
Yes. The relevant statistical tests and their results were reported for each figure.
We assumed normal distribution for all groups compared. However, this assumption was not tested manily because the sample size of each group (usually n<5 biological replicates) was too small.
Yes. In all quantitative results stanard errors (SEM, or SD) are reported to show the variation around the mean.
Litters from mice born a few days apart (our breeding cages are harems of one male and 2-3 females) were pooled and pups of similar weight were divided into vehicle and 144DG11 treated arms. Hence our studies were randomized.
In the in vivo experiments, as said, experimenters were blinded to arm (treated/vehicle) allocation.
In the cell-based and in vitro experiments, except for proteomics, experimenters were not blinded to group allocation. In the in silico experiments, group allocation is not applicable.
In all animal studies, experimenters were blinded to group allocation.

Data
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Sample size was demonstrated retrospectively to provide sufficient power based on the means and SD obtained. We required a sample size which would provide a power of at least 80% as calculated in Charan and Kantharia 2013.
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