Neuronal polyunsaturated fatty acids are protective in ALS/FTD

Ashling Giblin, Alexander J. Cammack, Niek Blomberg, Sharifah Anoar, Alla Mikheenko, Mireia Carcolé, Magda L. Atilano, Alex Hull, Dunxin Shen, Xiaoya Wei, Rachel Coneys, Lele Zhou, Yassene Mohammed, Damien Olivier-Jimenez, Lian Y. Wang, Kerri J. Kinghorn, Teresa Niccoli, Alyssa N. Coyne, Rik van der Kant, Tammaryn Lashley, Martin Giera, Linda Partridge, Adrian M. Isaacs
Nature Neuroscience
UK Dementia Research Institute, UCL, London, UK

Table of Contents

Overall Summary

Study Background and Main Findings

This research investigates the role of lipid metabolism in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases often linked by a mutation in the C9orf72 gene. The study's objective was to characterize changes in lipids (fats and fat-like substances) associated with this genetic mutation and to understand how these changes contribute to the death of nerve cells (neurodegeneration). The researchers used a multi-pronged approach, examining fruit flies (Drosophila) genetically engineered to mimic the disease, nerve cells grown in a lab from human stem cells (iPS cells), and brain tissue from deceased patients.

The study employed several key methods. "Transcriptomic analysis," specifically RNA sequencing, was used to measure the activity of genes involved in lipid metabolism. "Lipidomic analysis," using mass spectrometry, was used to identify and quantify the different types of lipids present in the various models. Genetic manipulation, including the overexpression of genes that increase the production of polyunsaturated fatty acids (PUFAs), and dietary supplementation with PUFAs were used to test the effects of altering lipid levels. Survival assays in fruit flies and measures of cell death in human cell cultures were used to assess the impact of these interventions.

The key finding was a consistent decrease in phospholipids (a major component of cell membranes) containing polyunsaturated fatty acids (PUFAs) across all the models studied: the C9orf72-mutant fruit flies, the human stem cell-derived neurons, and the postmortem brain tissue from FTD patients. Importantly, increasing PUFA levels, particularly within neurons, either through dietary supplementation or by genetically increasing the activity of enzymes that produce PUFAs (called desaturases), led to significant benefits. In the fruit fly model, this resulted in a 12-15% increase in median lifespan with dietary PUFA supplementation, and an even greater, though unquantified, increase when desaturase genes were overexpressed specifically in neurons. In human cell cultures, desaturase overexpression protected neurons from death induced by glutamate, a neurotransmitter that can become toxic at high levels (a process called excitotoxicity).

The study concludes that altered lipid metabolism, specifically a reduction in PUFA-containing phospholipids, plays a significant role in the development of ALS/FTD associated with the C9orf72 mutation. Furthermore, the research suggests that interventions aimed at increasing PUFA levels in neurons may offer a potential therapeutic strategy for these diseases.

Research Impact and Future Directions

This study provides compelling evidence for a link between altered lipid metabolism and the neurodegenerative diseases ALS and FTD, particularly those associated with the C9orf72 gene mutation. It's important to remember that correlation doesn't equal causation. While the study shows a strong association between reduced PUFA-containing phospholipids and disease progression, and interventions that increase PUFA levels show benefits, it doesn't definitively prove that the lipid changes cause the disease. Other factors could be at play, or the lipid changes could be a consequence of other underlying disease processes.

The practical implications of this research are significant. The findings suggest that targeting lipid metabolism, specifically by increasing neuronal PUFA levels, could be a viable therapeutic strategy for ALS/FTD. This is particularly relevant given the current lack of effective treatments for these devastating diseases. The study's use of multiple model systems, from fruit flies to human cells and postmortem tissue, strengthens the likelihood that these findings are relevant to human disease.

However, it's crucial to acknowledge the uncertainties. While the study demonstrates a beneficial effect of increasing PUFA levels, the optimal method for achieving this in humans, the long-term effects, and potential side effects remain unclear. Further research is needed to determine the best way to translate these findings into clinical practice. Dietary interventions, while promising, may not be sufficient to achieve the desired level of neuronal PUFA increase, as suggested by the greater benefit of direct genetic manipulation in the study.

Several key questions remain unanswered. The precise mechanism by which repeat RNA leads to reduced phospholipid desaturation needs further investigation. The study suggests potential roles for nucleocytoplasmic transport impairment and a 'lipid stress response,' but these hypotheses require further testing. Additionally, while the study focuses on neurons, the role of other brain cells, like glial cells, in lipid dysregulation and disease progression needs to be explored. The use of only one Drosophila G4C2 repeat line is a limitation, and future studies should investigate whether the number of repeats correlates with desaturation levels. Addressing these questions will be crucial for fully understanding the role of lipid metabolism in ALS/FTD and for developing effective therapeutic interventions.

Critical Analysis and Recommendations

Concise and Translational Summary (written-content)
The abstract concisely summarizes the core findings, linking transcriptomic (gene expression) and lipidomic (lipid profile) analyses across fruit flies, human stem cell-derived neurons, and human postmortem tissue. This multi-pronged approach strengthens the study's conclusions by demonstrating consistency across different biological systems. The clear statement of reduced PUFA-containing phospholipids and the beneficial effects of increasing neuronal PUFA levels provides a strong foundation for the study's significance.
Section: Abstract
Missing Disease Background (written-content)
The abstract does not provide context about ALS and FTD for a non-specialist reader. Including a brief introductory sentence about these diseases and their shared genetic basis (C9orf72 repeat expansion) would broaden the abstract's accessibility and impact, making it more understandable to a wider audience.
Section: Abstract
Effective Linking of ALS and FTD (written-content)
The introduction effectively establishes the link between ALS and FTD, describing them as two ends of a disease continuum with overlapping clinical, pathological, and genetic features. This provides a clear rationale for studying them together, justifying the combined investigation of these two related conditions.
Section: Introduction
Unclear Knowledge Gap (written-content)
The introduction does not explicitly state the knowledge gap regarding lipid metabolism's role in C9 ALS/FTD before this study. Adding a sentence or two outlining what was unknown before this research would clearly position the current work as directly addressing a significant unmet need in the field, strengthening the motivation for the study.
Section: Introduction
Conserved Gene Downregulation (written-content)
The study found a consistent downregulation of genes involved in fatty acid and lipid metabolism in both a Drosophila (fruit fly) model of C9orf72 repeat expansion and human postmortem spinal cord tissue. This cross-species validation, using RNA sequencing, suggests a fundamental and conserved role for lipid metabolism dysregulation in ALS/FTD pathology. This strengthens the potential relevance of findings to human disease.
Section: Results
Desaturase Overexpression Extends Survival (written-content)
Neuronal overexpression of fatty acid desaturase genes, particularly FAT-2 from C. elegans, significantly extended the survival of C9 Drosophila. Specifically, median survival increased by 12-15% with dietary PUFA supplementation, and a more substantial, though unquantified, extension was observed with neuronal desaturase overexpression. This demonstrates a causal link between neuronal lipid desaturation and neuroprotection in this model, suggesting a potential therapeutic target.
Section: Results
Improve Figure Referencing (written-content)
The Results section lacks a concise and direct link to the figures at the beginning of each subsection. While figures are referenced, making the connection more explicit and immediate (e.g., "RNA sequencing of Drosophila heads revealed... (Fig. 1a-d)") would guide the reader more effectively and improve the logical flow of the section.
Section: Results
Comprehensive Ethics Statement (written-content)
The Methods section provides a comprehensive ethics statement, covering both human iPS cell lines and postmortem brain tissue, demonstrating adherence to ethical guidelines and providing transparency. This is crucial for ethical research involving human subjects and enhances the credibility of the study.
Section: Methods
Insufficient iPS Cell Protocol Details (written-content)
The Methods section lacks sufficient detail regarding iPS cell culture and differentiation protocols. Key details such as specific media formulations, passage number, cell density, plating conditions, duration of differentiation stages, and confirmation of neuronal identity and purity are missing. This omission significantly hinders the reproducibility of the experiments, a cornerstone of scientific validity.
Section: Methods
Effective Summary of Main Findings (written-content)
The discussion effectively summarizes the study's main findings, highlighting the dysregulation of lipid metabolism in multiple C9 ALS/FTD models and the identification of a loss of PUFA-containing phospholipids. This provides a concise overview of the key results, reinforcing the core message of the study.
Section: Discussion
Acknowledgement of Limitation (written-content)
The discussion acknowledges a limitation of the study: the use of only one Drosophila G4C2 repeat line. This demonstrates scientific rigor and transparency, acknowledging potential constraints on the generalizability of the findings.
Section: Discussion
Lack of Specific Comparison to Literature (written-content)
The discussion does not delve into which specific lipids have been previously reported as altered in ALS/FTD and how the current findings compare. A more explicit comparison with existing literature on specific lipid alterations would strengthen the paper by demonstrating a deeper engagement with the existing body of knowledge and highlighting the novel contributions of the current study.
Section: Discussion

Section Analysis

Abstract

Key Aspects

Strengths

Suggestions for Improvement

Introduction

Key Aspects

Strengths

Suggestions for Improvement

Results

Key Aspects

Strengths

Suggestions for Improvement

Non-Text Elements

Fig. 1 | Transcriptomic and lipidomic analyses reveal downregulation of fatty...
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Fig. 1 | Transcriptomic and lipidomic analyses reveal downregulation of fatty acid and lipid metabolism genes and loss of PUFA-containing phospholipids in C9 flies.

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Fig. 1 | Transcriptomic and lipidomic analyses reveal downregulation of fatty acid and lipid metabolism genes and loss of PUFA-containing phospholipids in C9 flies.
First Reference in Text
To identify pathways dysregulated in neurons in response to expression of the pathological C9orf72 repeat (C9) expansion, we performed RNA sequencing (RNA-seq) on Drosophila heads with 36 G4C2 repeats expressed exclusively in adult neurons (Fig. 1a).
Description
  • The reference text describes RNA-seq experiments in Drosophila.: The reference text indicates that the figure provides data from RNA sequencing (RNA-seq) experiments. RNA-seq is a method used to figure out which genes are turned on or off in a cell or tissue. The experiment was performed on the heads of Drosophila (fruit flies). These flies had been genetically engineered to express 36 G4C2 repeats, which are a specific sequence of DNA. These repeats were only turned on in adult neurons, which are the cells in the brain that send electrical and chemical signals.
  • The reference text outlines the experiment's goal to identify dysregulated pathways.: The purpose of the experiment was to identify pathways that are dysregulated, meaning they are not working correctly, in neurons. This was done in response to the expression of the pathological C9orf72 repeat (C9) expansion. The C9orf72 repeat expansion is a mutation, a change in the DNA, that is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
Scientific Validity
  • The experimental design and methods are valid.: The approach is scientifically sound. RNA-seq is a well-established method for identifying changes in gene expression. Using Drosophila as a model organism is also a valid approach, as it allows for controlled genetic manipulation and relatively rapid experimentation. The use of adult neuron-specific expression of the C9orf72 repeat expansion ensures that the observed effects are relevant to the neuronal dysfunction seen in ALS/FTD.
  • The reference text includes key experimental parameters.: The reference text provides sufficient detail about the experimental design to assess its validity. The number of G4C2 repeats (36) is a relevant parameter, as the toxicity of the repeat expansion is thought to be related to the number of repeats. The restriction of expression to adult neurons is also important, as it avoids potential developmental effects.
Communication
  • The caption is clear and informative.: The caption provides a concise overview of the figure's content, effectively highlighting the key findings related to gene downregulation and phospholipid loss in the context of C9 flies. It clearly sets the stage for the more detailed information presented in the figure panels.
Fig. 2 | C9 repeats cause loss of highly unsaturated phospholipid species in...
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Fig. 2 | C9 repeats cause loss of highly unsaturated phospholipid species in iPS-cell-derived neurons.

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Fig. 2 | C9 repeats cause loss of highly unsaturated phospholipid species in iPS-cell-derived neurons.
First Reference in Text
As in the C9 flies, we observed a striking shift toward higher phospholipid saturation and loss of highly polyunsaturated phospholipids (containing fatty acyl chains with four or more double bonds) compared with controls (Fig. 2b, c and Extended Data Fig. 3a-c).
Description
  • The figure shows a shift in phospholipid saturation in iPS-cell-derived neurons.: The reference text indicates that the figure shows a shift towards higher phospholipid saturation and a loss of highly polyunsaturated phospholipids (PUFAs) in iPS-cell-derived neurons. Phospholipids are fats that are a critical component of cell membranes. Saturation refers to the number of double bonds in the fatty acid chains of the phospholipid. Saturated fats have no double bonds, while unsaturated fats have one or more. Polyunsaturated fats (PUFAs) are unsaturated fats with multiple double bonds. The change is observed when compared to control cells.
  • PUFAs are defined as having four or more double bonds.: The reference text specifies that highly polyunsaturated phospholipids are defined as those containing fatty acyl chains with four or more double bonds. Fatty acyl chains are the 'tails' of the phospholipid molecule, and the number of double bonds in these chains affects the fluidity and other physical properties of the cell membrane.
  • The findings are consistent with observations in C9 flies.: The reference text mentions that these findings are consistent with what was observed in the C9 flies. This suggests that the researchers are seeing a similar effect in both the fly model and the human cell model, strengthening the evidence that this is a real and important phenomenon.
Scientific Validity
  • The findings suggest a conserved mechanism across species.: The reference text suggests that the data presented in the figure provide evidence for a specific biochemical change (loss of PUFAs) in a relevant cell type (iPS-cell-derived neurons) in a model of C9-related disease. The consistency with findings in C9 flies strengthens the validity of the observation, suggesting a conserved mechanism across species.
  • The use of controls strengthens the validity.: The reference to specific figure panels (2b, c and Extended Data Fig. 3a-c) implies that these panels contain quantitative data supporting the claims made in the reference text. Without examining these panels, it's difficult to fully assess the statistical rigor and reproducibility of the findings. However, the mention of controls suggests that appropriate comparisons were made.
Communication
  • The caption is clear and concise.: The caption clearly states the main finding: C9 repeats, which are expansions of a specific DNA sequence, lead to a reduction in highly unsaturated phospholipids in neurons derived from induced pluripotent stem cells (iPS cells). This provides a concise summary of the figure's content and its relevance to the study.
Fig. 3 | Highly unsaturated phospholipids are decreased in FTLD postmortem...
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Fig. 3 | Highly unsaturated phospholipids are decreased in FTLD postmortem frontal cortex.

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Fig. 3 | Highly unsaturated phospholipids are decreased in FTLD postmortem frontal cortex.
First Reference in Text
In concordance with our fly and iPS cell-neuron data, in FTLD frontal cortex we observed a decrease in highly unsaturated phospholipids, particularly those containing four or more double bonds in their most unsaturated fatty acyl chain (Fig. 3b,c and Extended Data Fig. 6a,b), whereas lipid class proportionality was similar between control and FTLD tissue in both brain regions (Extended Data Fig.
Description
  • The figure shows decreased PUFAs in FTLD frontal cortex.: The reference text indicates that the researchers found a decrease in highly unsaturated phospholipids (PUFAs) in the frontal cortex of postmortem brains from individuals with frontotemporal lobar degeneration (FTLD). The brain samples were collected after the individuals had died (postmortem). PUFAs are a type of fat molecule found in cell membranes, and "highly unsaturated" means they contain multiple double bonds in their chemical structure. These double bonds make the membrane more fluid.
  • The decrease is most pronounced for phospholipids with 4+ double bonds.: The reference text specifies that the decrease was particularly noticeable for phospholipids containing four or more double bonds in their most unsaturated fatty acyl chain. Fatty acyl chains are the 'tails' of the phospholipid molecule, and the number of double bonds in these chains affects the fluidity and other physical properties of the cell membrane.
  • Lipid class proportionality is maintained.: The reference text notes that this finding is in concordance with their previous data from fly and iPS cell-neuron models, suggesting that this lipid alteration is relevant to the disease process across different models. The text also states that lipid class proportionality was similar between control and FTLD tissue. Lipid class proportionality means that the relative amounts of different types of lipids (e.g., phosphatidylcholine, phosphatidylethanolamine) were similar between the two groups.
Scientific Validity
  • The use of postmortem tissue is relevant but subject to confounds.: The use of postmortem human tissue is valuable for assessing disease-relevant changes in a realistic context. However, postmortem studies are subject to potential confounding factors, such as agonal state and postmortem interval, which should be carefully controlled for and reported. The concordance with fly and iPS cell-neuron data strengthens the validity of the finding, as it suggests that the observed lipid changes are not simply due to postmortem artifacts.
  • The use of controls and assessment of lipid class proportionality strengthens the validity.: The reference to specific figure panels (3b,c and Extended Data Fig. 6a,b) implies that these panels contain quantitative data supporting the claims made in the reference text. Without examining these panels, it's difficult to fully assess the statistical rigor and reproducibility of the findings. However, the mention of controls suggests that appropriate comparisons were made. The mention of lipid class proportionality being similar suggests that the overall lipid composition is not drastically altered, and the focus is on specific PUFA-containing species.
Communication
  • The caption is clear and direct.: The caption is direct and informative, clearly conveying the main finding of the figure: a reduction in highly unsaturated phospholipids in the frontal cortex of postmortem FTLD brains. This provides a clear expectation for what the figure will present.
Fig. 4 | Promoting fatty acid desaturation through either genetic or feeding...
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Fig. 4 | Promoting fatty acid desaturation through either genetic or feeding paradigms extends C9 fly survival and prevents cold-stress-induced death and paralysis.

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Fig. 4 | Promoting fatty acid desaturation through either genetic or feeding paradigms extends C9 fly survival and prevents cold-stress-induced death and paralysis.
First Reference in Text
(C18:2) and a-linolenic acid (C18:3) (Fig. 4a) significantly but modestly extended median survival of C9 flies by 12-15% (Fig. 4b, c and Extended Data Fig. 7a-c), whereas adding saturated or monounsaturated fatty acid species (palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1)) had either no effect or decreased survival (Extended Data Fig.
Description
  • PUFAs extended C9 fly survival by 12-15%.: The reference text states that linoleic acid (C18:2) and alpha-linolenic acid (C18:3) modestly extended the median survival of C9 flies by 12-15%. Linoleic acid and alpha-linolenic acid are polyunsaturated fatty acids (PUFAs). 'C18:2' and 'C18:3' refer to fatty acids with 18 carbon atoms and 2 or 3 double bonds, respectively. The median survival is the point at which 50% of the flies in a group are still alive.
  • Saturated/monounsaturated fats had no effect or decreased survival.: The reference text indicates that the addition of saturated or monounsaturated fatty acid species, like palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1), had either no effect or decreased survival. Saturated fatty acids have no double bonds, while monounsaturated fatty acids have one double bond.
  • The reference text specifies which figures present survival data.: Figure 4a is referenced, which likely shows a schematic of the fatty acid desaturation pathway. Figures 4b and 4c and Extended Data Fig. 7a-c are referenced as showing the survival data.
Scientific Validity
  • The use of controls and median survival strengthens the validity.: The use of median survival as a metric is appropriate for assessing the effects of dietary or genetic manipulations on lifespan. The 12-15% extension in median survival with PUFA supplementation, while modest, is statistically significant, suggesting a real effect. The use of saturated and monounsaturated fats as controls helps to determine the specificity of the PUFA effect.
  • The reference to specific data strengthens reproducibility.: Without examining the data in the referenced figures, it is difficult to assess the statistical rigor fully. However, the mention of specific fatty acids (C18:2, C18:3, C16:0, C18:0, C18:1) allows for precise replication of the experiment. The use of Extended Data Fig. suggests that more detailed data or controls are presented elsewhere.
Communication
  • The caption clearly summarizes the figure's content.: The caption is comprehensive, clearly indicating that the figure presents data on how promoting fatty acid desaturation, either by genetic manipulation or dietary intervention, affects the survival and stress resistance of C9 flies. It effectively sets the expectation for the specific types of experiments shown in the figure.
Fig. 5 FAT-1 and FAT-2 rescue glutamate-induced excitotoxicity in C9 and TDP-43...
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Fig. 5 FAT-1 and FAT-2 rescue glutamate-induced excitotoxicity in C9 and TDP-43 iPS cell-SNs.

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Fig. 5 FAT-1 and FAT-2 rescue glutamate-induced excitotoxicity in C9 and TDP-43 iPS cell-SNs.
First Reference in Text
We then tested whether overexpression of desaturase genes could prevent C9-associated neurodegeneration in human C9 neurons.
Description
  • The researchers tested whether desaturases prevent C9-associated neurodegeneration.: The reference text indicates that the researchers tested whether overexpressing desaturase genes could prevent neurodegeneration associated with C9 mutations in human C9 neurons. Desaturase genes encode enzymes that introduce double bonds into fatty acids, which is a process called desaturation. C9 mutations are expansions of a specific DNA sequence that are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • The experiment involved glutamate-induced excitotoxicity in iPS cell-derived spinal neurons.: Glutamate-induced excitotoxicity means that the researchers exposed the neurons to high levels of glutamate, a neurotransmitter that can become toxic at high concentrations, leading to cell death. iPS cell-SNs are spinal neurons derived from induced pluripotent stem cells, meaning that they are human cells that were reprogrammed to become stem cells and then differentiated into spinal cord neurons. Neurodegeneration is the progressive loss of structure or function of neurons.
Scientific Validity
  • The experimental design is scientifically sound.: The experimental design is scientifically sound. Testing the effect of desaturase overexpression on glutamate-induced excitotoxicity is a relevant approach for investigating neurodegeneration. Glutamate excitotoxicity is a well-established model for neuronal injury, and iPS cell-derived neurons are a useful tool for studying human disease.
  • The experiment is well-motivated and relevant.: The reference text provides a clear rationale for the experiment, as it builds on previous findings about lipid metabolism and neurodegeneration. The use of human C9 neurons is important, as it allows for direct assessment of the relevance of the findings to human disease.
Communication
  • The caption is clear and informative.: The caption is clear and informative. It explicitly states that the figure demonstrates the rescue of glutamate-induced excitotoxicity in C9 and TDP-43 iPS cell-derived spinal neurons (SNs) by FAT-1 and FAT-2, which are fatty acid desaturases.
Extended Data Fig. 1 | Fatty acid synthesis and desaturation pathway genes are...
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Extended Data Fig. 1 | Fatty acid synthesis and desaturation pathway genes are downregulated in C9 ALS post-mortem cervical spinal cord and C9 Drosophila.

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Extended Data Fig. 1 | Fatty acid synthesis and desaturation pathway genes are downregulated in C9 ALS post-mortem cervical spinal cord and C9 Drosophila.
First Reference in Text
Not explicitly referenced in main text
Description
  • The figure presents data on gene downregulation in C9 ALS models.: The extended data figure shows that genes involved in the creation (synthesis) and modification (desaturation) of fatty acids are less active (downregulated) in two different models of C9-ALS. C9-ALS refers to amyotrophic lateral sclerosis (ALS) caused by a mutation in the C9orf72 gene. One model is post-mortem cervical spinal cord, meaning tissue taken from the spinal cord of deceased individuals with C9-ALS. The cervical spinal cord is the part of the spinal cord in the neck region. The other model is C9 Drosophila, meaning fruit flies that have been genetically engineered to model C9-ALS.
  • The genes are involved in key lipid metabolism processes.: The genes are involved in fatty acid synthesis and desaturation. Fatty acid synthesis is the process by which cells make fatty acids from simpler building blocks. Desaturation is the process by which enzymes called desaturases add double bonds to fatty acids. The number of double bonds affects the properties of the fatty acid and the cell membrane.
Scientific Validity
  • The lack of explicit reference in the main text raises concerns.: The lack of explicit reference in the main text raises concerns about the figure's importance to the overall conclusions of the study. Extended data figures should provide essential supporting information that complements the main findings. If the figure is not referenced, it is unclear whether the data are critical to the study's conclusions.
  • The combination of human and Drosophila data strengthens the findings.: The combination of data from human tissue and a Drosophila model strengthens the validity of the findings, as it suggests that the observed gene downregulation is conserved across species. This provides stronger evidence that the observed changes are relevant to the disease process.
Communication
  • The caption is reasonably informative but could be more explicit.: The caption is reasonably informative, clearly stating that the figure presents data on the downregulation of genes involved in fatty acid synthesis and desaturation pathways. However, it could be more effective by explicitly mentioning that it combines data from two different models (human spinal cord and Drosophila) and the techniques used (e.g., RNA-seq, RT-qPCR).
Extended Data Fig. 2 | Lipidomic analyses in RNA-only (RO) and GR36 fly brains.
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Extended Data Fig. 2 | Lipidomic analyses in RNA-only (RO) and GR36 fly brains.
First Reference in Text
Not explicitly referenced in main text
Description
  • The figure presents lipidomic data from RO and GR36 fly brains.: The extended data figure presents lipidomic analyses, which involves measuring and identifying the different types of fats (lipids) present, in the brains of two types of fruit flies: RNA-only (RO) and GR36. These flies are genetically modified strains used to study the effects of the C9orf72 repeat expansion, which is a mutation linked to ALS and FTD. RO flies have a repeat sequence that is interrupted by stop codons, preventing the production of dipeptide repeat proteins (DPRs). GR36 flies produce toxic poly(GR) through ATG-driven translation.
Scientific Validity
  • The lack of explicit reference makes it difficult to assess the importance of this figure.: The lack of explicit reference in the main text makes it difficult to assess the importance of this figure. The RO and GR36 fly models are used to disentangle the effects of repeat RNA and DPRs. Lipidomic analyses are useful for determining whether the lipid profile is altered in these models. Without knowing the specific hypothesis being tested with these data, it is difficult to judge its scientific validity.
  • The data likely provide supporting evidence but are not essential to the core findings.: The inclusion of these data in an extended data figure suggests that they provide supporting evidence for a claim made in the main text, but are not essential for understanding the core findings. The data may help to rule out alternative explanations or provide additional details about the lipid changes observed in the different fly models.
Communication
  • The caption is clear but lacks context without referring to the main text.: The caption clearly identifies the figure as presenting lipidomic analyses, which means the measurement of all the lipids in a sample. It also specifies that the analyses were performed on the brains of two different types of fruit flies: RNA-only (RO) and GR36. However, the significance of these fly types is not immediately clear from the caption alone, requiring the reader to refer to the main text or other figures for context.
Extended Data Fig. 3 | Phospholipid levels in i³Neurons, displayed as separate...
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Extended Data Fig. 3 | Phospholipid levels in i³Neurons, displayed as separate neuronal inductions/lines.

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Extended Data Fig. 3 | Phospholipid levels in i³Neurons, displayed as separate neuronal inductions/lines.
First Reference in Text
As in the C9 flies, we observed a striking shift toward higher phospholipid saturation and loss of highly polyunsaturated phospholipids (containing fatty acyl chains with four or more double bonds) compared with controls (Fig. 2b, c and Extended Data Fig. 3a-c).
Description
  • The figure presents phospholipid levels in i³Neurons, displayed separately for each neuronal induction/line.: The extended data figure presents phospholipid levels in i³Neurons. i³Neurons are cortical neurons derived from induced pluripotent stem cells (iPSCs) using a specific protocol. The data are displayed as separate neuronal inductions/lines, meaning that each line represents an independent experiment where iPSCs were differentiated into neurons. This allows for assessing the reproducibility of the results across different experimental runs.
  • The data relate to the main finding of a shift toward higher phospholipid saturation and loss of PUFAs.: The reference text connects this figure to the main finding of a shift toward higher phospholipid saturation and loss of highly polyunsaturated phospholipids (PUFAs), as observed in C9 flies. This suggests that the data in this extended data figure provide further support for this finding in the i³Neuron model.
Scientific Validity
  • Displaying separate inductions/lines strengthens the validity.: Displaying the data for separate neuronal inductions/lines is a strength, as it allows the reader to assess the reproducibility of the findings across independent experiments. This increases confidence in the robustness of the observed effects. The connection to the main finding regarding phospholipid saturation and PUFA loss suggests that this figure provides important supporting evidence.
  • The statistical rigor cannot be fully assessed without examining the figure.: Without examining the figure itself, it is difficult to fully assess the statistical rigor. It is important that appropriate statistical tests were used to compare phospholipid levels between different conditions, and that the results of these tests are clearly presented in the figure legend or accompanying text.
Communication
  • The caption is informative but could benefit from stating the purpose of the data presentation.: The caption provides essential information about the figure's content, specifying that it shows phospholipid levels in i³Neurons (a specific type of induced neuron) and that the data are presented for separate neuronal inductions/lines. This is helpful for understanding the structure and interpretation of the data. However, the caption could be enhanced by briefly mentioning the purpose of showing the data in this way (e.g., to demonstrate reproducibility across different inductions).
Extended Data Fig. 4 | Poly(GA) levels in i³Neuron lines treated with (G4C2)...
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Extended Data Fig. 4 | Poly(GA) levels in i³Neuron lines treated with (G4C2) lentiviruses or sense repeat-targeted antisense oligonucleotides (ASOs).

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