Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14)
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Abstract
Infantile neuroaxonal dystrophy (INAD) is caused by recessive variants in PLA2G6 and is a lethal pediatric neurodegenerative disorder. Loss of the Drosophila homolog of PLA2G6 , leads to ceramide accumulation, lysosome expansion, and mitochondrial defects. Here, we report that retromer function, ceramide metabolism, the endolysosomal pathway, and mitochondrial morphology are affected in INAD patient-derived neurons. We show that in INAD mouse models, the same features are affected in Purkinje cells, arguing that the neuropathological mechanisms are evolutionary conserved and that these features can be used as biomarkers. We tested 20 drugs that target these pathways and found that Ambroxol, Desipramine, Azoramide, and Genistein alleviate neurodegenerative phenotypes in INAD flies and INAD patient-derived neural progenitor cells. We also develop an AAV-based gene therapy approach that delays neurodegeneration and prolongs lifespan in an INAD mouse model.
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Author Response
Reviewer #1 (Public Review):
Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patientderived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs …
Author Response
Reviewer #1 (Public Review):
Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patientderived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs that express human PLA2G6 and study expression levels in a human kidney cell line as well as in patent-derived NPCs. In the latter neuronal model, they show that expression of human PLA2G6 can rescue mitochondrial fragmentation associated with PLA2G6 loss-of-function. Lin et al then show that ICV (intracerebroventricular) and IV (intravenous) injection of a human PLA2G6-containing construct is able to partially rescue the rotarod phenotype in PLA2G6 transheterozygous PLA2G6 mutant mice between ~110 and 150 days. There is also an associated improvement in lifespan and body weight.
The strengths of this work are that the authors use a number of different model organism systems, including patient-derived neuronal cells, Drosophila models (INAD flies) and mouse models to study PLA2G6-associated neurodegeneration (PLAN) at the cellular level. They also screen drug compounds that are predicted to target endo-lysosomal trafficking and sphingolipid metabolic pathways to ameliorate PLAN, thus identifying potential new therapeutic strategies. The work in mice, showing that gene therapy with human PLA2G6 can rescue a behavioural phenotype and lifespan is the first proof-ofconcept of such an advancement. This work will hopefully lead to further studies for optimisation toward clinical advancement.
We thank the reviewer and editor for the positive comments about our manuscript.
The major weaknesses are that the pathogenic mechanisms shown in the patient-derived neuronal cells and mice do not extend as far as those previously shown in the fly model published by the authors. Of note, ceramide levels and retromer function are not studied, both key pathologies described in the previous fly models. In addition, the drug screening is limited by its testing in one fly behavioural assay and LAMP2 Western blot analysis on patient derived NPCs.
The results, in general, support the conclusions of the authors and represent well-performed work. However, the significance of elevated glucosylceramide levels is not clear in the present study. Although this was previously found to be elevated in INAD flies, it was ceramide levels that were thought to be the main toxic insult, with drugs aimed at reducing ceramide levels being shown to rescue INAD flies.
We addressed these concerns. Please refer to our response to each of the specific point listed below.
This work will no doubt be of significant interest to the field, confirming several previous findings in the Drosophila model of PLA2G6 (iPLA2-VIA) knockout. It also extends upon the fly work by identifying compounds that can be further studied for potential drug-re-purposing for the treatment of PLA2G6associated disease. The gene therapy studies are also very interesting and a first proof-of-principle in PLAN using ICV and IV delivery in a mouse model.
We thank the reviewers and editor as addressing all these concerns really improved the manuscript.
Reviewer #2 (Public Review):
This article aims to extend human disease-related studies of PLA2G6 from fly models to iPS-neurons, mouse models, to look for drugs that suppress phenotypes and test them, and to attempt AAV whole body rescue. Generally, each of these questions/aims/experiments is excellent, but as presented, it's a bit of an underdeveloped hodgepodge of results, with each experiment somewhat underdeveloped or analyzed for the respective phenotype, in my opinion. I think the general thrust of the experiments is excellent. But the data are relatively cursory in many instances. Further development and characterization of the phenotypes would require quite a bit of work but vastly improve the paper.
We thank the reviewer for the positive comments about our manuscript. We have addressed most of the concerns.
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eLife assessment
This work is of significant interest to those studying neurodegeneration, demonstrating key pathologies in PLA2G6-associated disease in both patient-derived neuronal models and a novel trans heterozygote mouse model. Moreover, it identifies a number of possible compounds that could potentially be re-purposed for therapeutic use in PLA2G6-associated neurodegeneration. Lastly, it shows a proof-of-principle in a mouse model that gene therapy with human PLA2G6 can rescue defects in PLA2G6 deficiency. Whilst the majority of the data are solid and convincing, there are a number of consolidatory experiments that would add greatly to the overall impact and novelty of the work.
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Reviewer #1 (Public Review):
Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patient-derived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs that express human …
Reviewer #1 (Public Review):
Lin et al. characterise cellular pathologies in PLA2G6 mutant patient-derived neuronal cells (neuronal progenitor cells, NPCs, and IPSc-derived dopaminergic neurones) and a novel compound heterozygous PLA2G6 mutant mouse model. They build on their previous findings in an INAD fly model (lacking PLA2G6) to show that lysosomal and mitochondrial defects are evolutionary conserved in PLA2G6 deficiency. The authors proceed to use their INAD fly model and to screen a number of compounds that are predicted to modulate endo-lysosomal function using a bang sensitivity assay. They then show that the drugs that can rescue this fly behavioural phenotype also reduce LAMP2 expression in patient-derived NPCs on Western blot analysis. Lastly, the manuscript reports the creation of new genetic constructs that express human PLA2G6 and study expression levels in a human kidney cell line as well as in patent-derived NPCs. In the latter neuronal model, they show that expression of human PLA2G6 can rescue mitochondrial fragmentation associated with PLA2G6 loss-of-function. Lin et al then show that ICV (intracerebroventricular) and IV (intravenous) injection of a human PLA2G6-containing construct is able to partially rescue the rotarod phenotype in PLA2G6 transheterozygous PLA2G6 mutant mice between ~110 and 150 days. There is also an associated improvement in lifespan and body weight.
The strengths of this work are that the authors use a number of different model organism systems, including patient-derived neuronal cells, Drosophila models (INAD flies) and mouse models to study PLA2G6-associated neurodegeneration (PLAN) at the cellular level. They also screen drug compounds that are predicted to target endo-lysosomal trafficking and sphingolipid metabolic pathways to ameliorate PLAN, thus identifying potential new therapeutic strategies. The work in mice, showing that gene therapy with human PLA2G6 can rescue a behavioural phenotype and lifespan is the first proof-of-concept of such an advancement. This work will hopefully lead to further studies for optimisation toward clinical advancement.
The major weaknesses are that the pathogenic mechanisms shown in the patient-derived neuronal cells and mice do not extend as far as those previously shown in the fly model published by the authors. Of note, ceramide levels and retromer function are not studied, both key pathologies described in the previous fly models. In addition, the drug screening is limited by its testing in one fly behavioural assay and LAMP2 Western blot analysis on patient derived NPCs.
The results, in general, support the conclusions of the authors and represent well-performed work. However, the significance of elevated glucosylceramide levels is not clear in the present study. Although this was previously found to be elevated in INAD flies, it was ceramide levels that were thought to be the main toxic insult, with drugs aimed at reducing ceramide levels being shown to rescue INAD flies.
This work will no doubt be of significant interest to the field, confirming several previous findings in the Drosophila model of PLA2G6 (iPLA2-VIA) knockout. It also extends upon the fly work by identifying compounds that can be further studied for potential drug-re-purposing for the treatment of PLA2G6-associated disease. The gene therapy studies are also very interesting and a first proof-of-principle in PLAN using ICV and IV delivery in a mouse model.
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