Glial ferritin maintains neural stem cells via transporting iron required for self-renewal in Drosophila

Reviewer #1 (Public Review):

Summary:
This study unveils a novel role for ferritin in Drosophila larval brain development. Furthermore, it pinpoints that the observed defects in larval brain development resulting from ferritin knockdown are attributed to impaired Fe-S cluster activity and ATP production. In addition, knocking down ferritin genes suppressed the formation of brain tumors induced by brat or numb RNAi in Drosophila larval brains. Similarly, iron deficiency suppressed glioma in the mice model. Overall, this is a well-conducted and novel study.

Strengths:
Thorough analyses with the elucidation of molecular mechanisms.

Weaknesses:
Some of the conclusions are not well supported by the results presented.

Reviewer #2 (Public Review):

Summary:
Zhixin and collaborators have investigated if the molecular pathways present in glia play a role in the proliferation, maintenance, and differentiation of Neural Stem Cells. In this case, Drosophila Neuroblasts are used as models. The authors find that neuronal iron metabolism modulated by glial ferritin is an essential element for Neuroblast proliferation and differentiation. They show that loss of glial ferritin is sufficient to impact on the number of neuroblasts. Remarkably, the authors have identified that ferritin produced in the glia is secreted to be used as an iron source by the neurons. Therefore iron defects in glia have serious consequences in neuroblasts and likely vice versa. Interestingly, preventing iron absorption in the intestine is sufficient to reduce NB number. Furthermore, they have identified Zip13 as another regulator of the process. The evidence presented strongly indicates that loss of neuroblasts is due to premature differentiation rather than cell death.

Strengths:
- Comprenhensive analysis of the impact of glial iron metabolism in neuroblast behaviour by genetic and drug-based approaches as well as using a second model (mouse) for some validations.
- Using cutting-edge methods such as RNAseq as well as very elegant and clean approaches such as RNAi-resistant lines or temperature-sensitive tools
- Goes beyond the state of the art highlighting iron as a key element in neuroblast formation as well as as a target in tumor treatments.

Weaknesses:
Although the manuscripts have clear strengths, there are also some strong weaknesses that need to be addressed.
- Some literature is missing
- In general, the authors succeeded but in some cases, the authors´ claims are not fully supported by the evidence presented and additional experiments are critical to discriminate among different hypotheses.
- Moreover, some potential flaws might be present in the analysis of cell death and mitochondrial iron.

Reviewer #3 (Public Review):

In this manuscript, Ma et al seek to identify stem cell niche factors. They perform an RNAi screen in glial cells and screen for candidates that support and maintain neuroblasts (NBs) in the developing fly brain. Through this, they identify two subunits of ferritin, which is a conserved protein that can store iron in cells in a non-toxic form and release it in a controlled manner when and where required. They present data to support the conclusion that ferritin produced in glia is released and taken up by NBs where it is utilised by enzymes in the Krebs cycle as well as in the electron transport chain. In its absence from glia, NBs are unable to generate sufficient energy for division and therefore prematurely differentiate via nuclear prospero resulting in small brains. The work will be of interest to those interested in neural stem cells and their non-cell autonomous control by niches.

The past decade has seen a growing appreciation of how glial cells support and maintain NBs during development. The authors' discovery of glial-derived ferritin providing essential iron atoms for energy production is interesting and important. They have employed a variety of genetic tools and assays to uncover how ferritin in glia might support NBs. This is particularly challenging because there are no direct ways of assaying for iron or energy consumption in a cell-specific manner.

There are however instances where conclusions are drawn to support the story being developed without considering the equally plausible alternative explanations that should ideally be addressed.

For example, the data supporting the transfer of ferritin from glia to NBs was weak given the misexpression system used; the Shi[ts] experiment was also not convincing (perhaps they have more representative images?).

The iron manipulation experiments are in the whole animal and it is likely that this affects general feeding behaviour, which is known to affect NB exit from quiescence and proliferative capacity. The loss of ferritin in the gut and iron chelators enhancing the NB phenotype are used as evidence that glia provide iron to NB to support their number and proliferation. Since the loss of NB is a phenotype that could result from many possible underlying causes (including low nutrition), this specific conclusion is one of many possibilities.

Similarly, knockdown of the FeS protein assembly components phenocopy glial ferritin knock down. Since iron is so important for the TCA and the ETC, this is not surprising, but the similarities in the two phenotypes seem insufficient to say that it's glial ferritin that's causing the lack of iron in the NB and therefore resulting in loss of NBs.

Pros RNAi will certainly result in an increase in NB numbers because the loss of pros results in an inability of NB progeny to differentiate. This (despite the slight increase in nuclear pros) is not sufficient to infer that glial ferritin knockdown results in premature differentiation of NBs via nuclear pros.

I recognise these are challenging to prove irrefutably, however, the frequency of such expansive interpretations of data is of concern.

Author Response

eLife assessment

This study, which seeks to identify factors from the glial niche that support and maintain neural stem cells, unveils a novel role for ferritin in this process. Furthermore, the work shows that defects in larval brain development resulting from ferritin knockdown can be attributed to impaired Fe-S cluster activity and ATP production. These findings will be valuable to both oncologists and neurobiologists, though the supporting evidence is currently incomplete.

Public Reviews

Reviewer #1 (Public Review):

Summary:

This study unveils a novel role for ferritin in Drosophila larval brain development. Furthermore, it pinpoints that the observed defects in larval brain development resulting from ferritin knockdown are attributed to impaired Fe-S cluster activity and ATP production. In addition, knocking down ferritin genes suppressed the formation of brain tumors induced by brat or numb RNAi in Drosophila larval brains. Similarly, iron deficiency suppressed glioma in the mice model. Overall, this is a well-conducted and novel study.

Strengths:

Thorough analyses with the elucidation of molecular mechanisms.

Weaknesses:

Some of the conclusions are not well supported by the results presented.

We really appreciate your review and positive feedback. As for weaknesses, we will try our best to solidate the related conclusions.

Reviewer #2 (Public Review):

Summary:

Zhixin and collaborators have investigated if the molecular pathways present in glia play a role in the proliferation, maintenance, and differentiation of Neural Stem Cells. In this case, Drosophila Neuroblasts are used as models. The authors find that neuronal iron metabolism modulated by glial ferritin is an essential element for Neuroblast proliferation and differentiation. They show that loss of glial ferritin is sufficient to impact on the number of neuroblasts. Remarkably, the authors have identified that ferritin produced in the glia is secreted to be used as an iron source by the neurons. Therefore iron defects in glia have serious consequences in neuroblasts and likely vice versa. Interestingly, preventing iron absorption in the intestine is sufficient to reduce NB number. Furthermore, they have identified Zip13 as another regulator of the process. The evidence presented strongly indicates that loss of neuroblasts is due to premature differentiation rather than cell death.

Strengths:

• Comprenhensive analysis of the impact of glial iron metabolism in neuroblast behaviour by genetic and drug-based approaches as well as using a second model (mouse) for some validations.

• Using cutting-edge methods such as RNAseq as well as very elegant and clean approaches such as RNAi-resistant lines or temperature-sensitive tools

• Goes beyond the state of the art highlighting iron as a key element in neuroblast formation as well as as a target in tumor treatments.

Weaknesses:

Although the manuscripts have clear strengths, there are also some strong weaknesses that need to be addressed.

• Some literature is missing

Thanks for your reminder and we will add the missing literatures.

• In general, the authors succeeded but in some cases, the authors´ claims are not fully supported by the evidence presented and additional experiments are critical to discriminate among different hypotheses.

We are greatly grateful to the reviewer for recognizing our work, and we will support our conclusions with further evidence.

• Moreover, some potential flaws might be present in the analysis of cell death and mitochondrial iron.

We used Caspase-3 or TUNEL to indicate the apoptosis signal. Further, we overexpressed the anti-apoptosis gene p35 to inhibit apoptosis and found no rescue effect on neuroblast number. The results of these experiments are consistent.

It is difficult to determine the mitochondrial iron of neuroblast, so we used indirect methods to test ferroptosis, such as TEM and iron (or iron chelator) supplement. We will perform more experiments according to recommendations to determine that.

Reviewer #3 (Public Review):

In this manuscript, Ma et al seek to identify stem cell niche factors. They perform an RNAi screen in glial cells and screen for candidates that support and maintain neuroblasts (NBs) in the developing fly brain. Through this, they identify two subunits of ferritin, which is a conserved protein that can store iron in cells in a non-toxic form and release it in a controlled manner when and where required. They present data to support the conclusion that ferritin produced in glia is released and taken up by NBs where it is utilised by enzymes in the Krebs cycle as well as in the electron transport chain. In its absence from glia, NBs are unable to generate sufficient energy for division and therefore prematurely differentiate via nuclear prospero resulting in small brains. The work will be of interest to those interested in neural stem cells and their non-cell autonomous control by niches.

The past decade has seen a growing appreciation of how glial cells support and maintain NBs during development.

The authors' discovery of glial-derived ferritin providing essential iron atoms for energy production is interesting and important. They have employed a variety of genetic tools and assays to uncover how ferritin in glia might support NBs. This is particularly challenging because there are no direct ways of assaying for iron or energy consumption in a cell-specific manner.

There are however instances where conclusions are drawn to support the story being developed without considering the equally plausible alternative explanations that should ideally be addressed.

For example, the data supporting the transfer of ferritin from glia to NBs was weak given the misexpression system used; the Shi[ts] experiment was also not convincing (perhaps they have more representative images?).

Thanks for your comment. We have the negative control, which excludes the misexpression. As for Shits experiment, we will substitute for more representative images.

The iron manipulation experiments are in the whole animal and it is likely that this affects general feeding behaviour, which is known to affect NB exit from quiescence and proliferative capacity. The loss of ferritin in the gut and iron chelators enhancing the NB phenotype are used as evidence that glia provide iron to NB to support their number and proliferation. Since the loss of NB is a phenotype that could result from many possible underlying causes (including low nutrition), this specific conclusion is one of many possibilities.

Iron chelator (or iron salt) feeding is a common method for investigating metal metabolism in Drosophila[1-3]. And other metal chelators (such as copper and zinc chelator) do not have similar phenotype (data not shown), which can partially exclude this possibility. Further, iron absorption was blocked by knockdown of ferritin only in the iron cell region[1], a small part of midgut, which phenocopied iron chelator feeding, implying iron deficiency is probably the main cause of the phenotype. More importantly, iron chelator only enhances the NB phenotype in the ferritin knockdown group, not the control group, suggesting iron deficiency results in the phenotype, which rules out other possibilities.

Similarly, knockdown of the FeS protein assembly components phenocopy glial ferritin knock down. Since iron is so important for the TCA and the ETC, this is not surprising, but the similarities in the two phenotypes seem insufficient to say that it's glial ferritin that's causing the lack of iron in the NB and therefore resulting in loss of NBs.

It is hard to get this conclusion just by FeS protein assembly components knockdown, so we just used “implied” to describe this result. However, we combine several results to address this issue, including iron chelator feeding, ferritin knockdown in the midgut, the enhancement of phenotype by iron chelators, aconitase activity, GO enrichment, KEGG enrichment, and Zip13. These results pointed to the interpretation that iron deficiency in NBs caused by glial ferritin defects leads to NB loss.

Pros RNAi will certainly result in an increase in NB numbers because the loss of pros results in an inability of NB progeny to differentiate. This (despite the slight increase in nuclear pros) is not sufficient to infer that glial ferritin knockdown results in premature differentiation of NBs via nuclear pros.

First, pros RNAi, brat RNAi, or numb RNAi can each result in an inability of NB progeny to differentiate, respectively[4-6]. If the rescue of NB number by pros RNAi mainly relies on the differentiation block of NB progeny, brat RNAi or numb RNAi is expected to similarly rescue the NB number. However, our results showed that only pros RNAi could rescue the NB number, while brat RNAi or numb RNAi could not.

Secondly, nuclear Pros represses genes required for self-renewal and is also required to activate genes for terminal differentiation[7]. Thus, Pros is kept in the cytoplasm and remains almost undetectable in the nuclei in normal NBs[8]. However, we observed the detectable Pros in the nuclei of some NBs after glial ferritin knockdown, and the NB number with detectable nuclear Pros was significantly increased when compared to control.

Altogether, we conclude that NBs tend to undergo premature differentiation after glial ferritin knockdown.

I recognise these are challenging to prove irrefutably, however, the frequency of such expansive interpretations of data is of concern.

(1) Tang X, Zhou B. Ferritin is the key to dietary iron absorption and tissue iron detoxification in Drosophila melanogaster. FASEB J, 2013,27(1):288-98

(2) Xiao G, Liu ZH, Zhao M, et al. Transferrin 1 Functions in Iron Trafficking and Genetically Interacts with Ferritin in Drosophila melanogaster. Cell Rep, 2019,26(3):748-58 e5

(3) Mukherjee C, Kling T, Russo B, et al. Oligodendrocytes Provide Antioxidant Defense Function for Neurons by Secreting Ferritin Heavy Chain. Cell Metab, 2020,32(2):259-72 e10

(4) Knoblich JA, Jan LY, Jan YN. Asymmetric Segregation of Numb and Prospero during Cell-Division. Nature, 1995,377(6550):624-7

(5) Zacharioudaki E, Magadi SS, Delidakis C. bHLH-O proteins are crucial for neuroblast self-renewal and mediate Notch-induced overproliferation. Development, 2012,139(7):1258-69

(6) Bello B, Reichert H, Hirth F. The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of. Development, 2006,133(14):2639-48

(7) Choksi SP, Southall TD, Bossing T, et al. Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Developmental Cell, 2006,11(6):775-89

(8) Spana EP, Doe CQ. The Prospero Transcription Factor Is Asymmetrically Localized to the Cell Cortex during Neuroblast Mitosis in Drosophila. Development, 1995,121(10):3187-95