Ggnbp2 regulates synaptic development and autophagy in motor neurons
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Abstract
Genome-wide association studies (GWAS) have identified numerous candidate ALS risk variants, but their cellular functions are often unknown. Recent studies have identified a variant of GGNBP2 that results in increased expression. To better understand how this gene might contribute to disease, we investigated the function of Drosophila Ggnbp2 (dGgnbp2) in motor neurons. Loss of function studies showed that dGgnbp2 is required for motor neuron synaptic development. A human transgene completely rescued these phenotypes indicating that the gene is functionally conserved between humans and flies. Overexpression of dGgnbp2 caused severe locomotor defects in adult flies, consistent with ALS pathology. At the cellular level, dGgnbp2 regulated autophagy, a process commonly defective in ALS. Both overexpression and removal of dGgnbp2 reduced levels of the phosphorylated lipid, PI(3)P, an essential component of autophagosomes. Our study provides strong evidence that Ggnbp2 functions in motor neurons to regulate a cellular process commonly defective in ALS.
Teaser
This study investigated the function of the ALS risk variant GGNBP2 , in flies, and showed that it regulates autophagy in motor neurons.
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Referee #3
Evidence, reproducibility and clarity
Genome-wide association studies (GWAS) were used to identify potential risk variants associated with amyotrophic lateral sclerosis (ALS), and a specific variant of GGNBP2 has emerged as a critical player, exhibiting increased expression, and prompting the authors to investigate its role in the disease progression.
The study, focusing on Drosophila Ggnbp2 (dGgnbp2) in motor neurons, revealed crucial insights into its function. Loss-of-function experiments underscored the necessity of dGgnbp2 in motor neuron synaptic development. Strikingly, introducing a human transgene fully restored these phenotypes, demonstrating functional …
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Referee #3
Evidence, reproducibility and clarity
Genome-wide association studies (GWAS) were used to identify potential risk variants associated with amyotrophic lateral sclerosis (ALS), and a specific variant of GGNBP2 has emerged as a critical player, exhibiting increased expression, and prompting the authors to investigate its role in the disease progression.
The study, focusing on Drosophila Ggnbp2 (dGgnbp2) in motor neurons, revealed crucial insights into its function. Loss-of-function experiments underscored the necessity of dGgnbp2 in motor neuron synaptic development. Strikingly, introducing a human transgene fully restored these phenotypes, demonstrating functional conservation between humans and flies. Contrary to expectations, overexpression of dGgnbp2 resulted in severe locomotor defects in adult flies, mirroring aspects of ALS pathology, suggesting a tight regulation for this protein in the control of NMJ function.
The authors also suggest a role of the gene in regulating autophagy, as RNA-seq showed abnormalities in the expression levels of genes involved in autophagy when dGGNPB2 was mutated. Finally, they described a potential molecular mechanism through which GGNBP2 may regulate autophagy and that is by controlling phospholipid levels specifically PI(3)P.
Understanding the cellular mechanisms, the study suggests dGgnbp2's role in regulating autophagy, a process frequently impaired in ALS, and both overexpression and depletion of dGgnbp2 led to altered levels of phosphorylated lipid PI(3)P, a vital component of autophagosomes. Add PI3K This comprehensive investigation provides compelling evidence that Ggnbp2 plays a pivotal role in motor neurons, exerting regulatory control over a cellular process commonly compromised in ALS. These findings also provide insights into the functional implications of the GGNBP2 variant and also open new avenues for potential therapeutic crucial pathways like autophagy and InR, offering a promising direction in ALS treatment.
Major:
The experiments outlined in this paper, which elucidate the function of the Ggnbp2 in the neuromuscular junction (NMJ), are compelling and crucial for characterizing a novel gene implicated in ALS. However, my primary concern revolves around the demonstration of the influential role of the Ggnbp2 in regulating autophagy and its responsiveness to insulin signaling.
Indeed, the author should better analyze and also genetically demonstrate an interaction between Ggnbp2 and components of the insulin signaling pathway. For instance, analyzing PTEN (utilizing available mutants) could enhance the understanding of the pathway linking Ggnbp2 downstream of PIP2.
The connections drawn in the paragraph regarding autophagy (paragraph title: dGgnbp2 is linked to autophagy in motor neurons) might be a bit tenuous for the described observations. In the end it seems to only suggest a link to autophagy without explicitly asserting that it drives autophagy. The authors have overlooked providing a clear explanation or speculation on the correlation between GGNBP2's regulation of autophagy and its impact on synaptic development. A more explicit emphasis, then, on whether a loss-of-function or gain-of-function is more pertinent to human disease is necessary.
The authors should take advantage of the available Drosophila lines to elucidate the relative dependence of the autophagic flux controlled by Ggnbp2 and macroautophagy, using mutants or RNAi lines for Atg1, Atg5, and potentially Atg6/beclin, as these factors have been demonstrated to be relevant in neurodegenerative diseases, including ALS and Parkinson's.
Furthermore, alternative approaches for testing InR activation exist, such as the widely employed tPH-GFP method (Britton et al., 2002) that could be used to implement the activation of Akt in NMJ.
The observation that dGgnbp2 serves specific functions in the cytoplasm of motor neurons is particularly interesting, and further investigation to better understand this function would be valuable. Additionally, is this soluble form also identified in humans?
Minor:
Line 113-119: This does not seem relevant to the study. This is not discussed or investigated anywhere else in this work. However, I understand and appreciate the intent.
Line 135-138/Figure 2L: The authors do not address the non-significant "continuous" data.
Line 191: Define the age of young animals.
Line 192: Why did the authors change to OK371-Gal4 promoter in the experiments in Figure 4.
Line 192: The authors need to specify how young were the animals.
Line 202: Is the human GGNBP2 expressed with OK6-Gal4 able to rescue the reduced motility of the d Ggnbp2null flies?
Line 223: Typo, "to the" is repeated.
Referees cross-commenting
The comments I read are all feasible and in line with what I also suggest, and I accept them. What is not clear is how much time the authors need if they reply to all, which, of course, depends on how much deeper they decide to go to complete the characterization of GGNBP2 in autophagy and the relevance of InR/TOR signaling. This will also depend on the journal they decide for their final submission.
Significance
This study outlined in this paper rests on a robust experimental design and yields conclusive results. It shows strong evidence of a role of the GGNBP2 gene in ALS pathology. It provides clear evidence of a synaptic development defect as well as a locomotor dysfunction in mutants, building on its relevance in human pathology. It solidly proves a functional conservation between flies and humans. It then suggests a potential role in regulating autophagy. However, further experiments demonstrating the actual interaction between GGNBP2 and class I and III PI3K is needed to fully elucidate the mechanisms through which GGNBP2 controls autophagy.
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Referee #2
Evidence, reproducibility and clarity
Kerwin et al. conducted a study on the function of dGgnbp2 and its role in motoneurons. There are some general oversights that make the paper less robust than it could be. Firstly, the connection between autophagy dysregulation and the autophagic defects shown in the last figures is not clear. Combining these two parts would add more value to the paper. Secondly, the actual connection to ALS - made by the authors throughout the study - is not very strong. The only link with ALS is mentioned at the beginning when it is stated that Futsch is a target of TDP-43 and later when the authors mention that autophagy is a key dysregulated …
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Referee #2
Evidence, reproducibility and clarity
Kerwin et al. conducted a study on the function of dGgnbp2 and its role in motoneurons. There are some general oversights that make the paper less robust than it could be. Firstly, the connection between autophagy dysregulation and the autophagic defects shown in the last figures is not clear. Combining these two parts would add more value to the paper. Secondly, the actual connection to ALS - made by the authors throughout the study - is not very strong. The only link with ALS is mentioned at the beginning when it is stated that Futsch is a target of TDP-43 and later when the authors mention that autophagy is a key dysregulated pathway in ALS. Although this is true, there is no strong connection to ALS pathology. It would have been appropriate to measure TDP-43 mislocalization in the animals and see whether the Futsch decrease observed in both null and OE models is related to it, or whether TDP-43 aggregates.
Figure 2: Although the results presented in Fig S3R-X suggest a possible reduction in dGgnbp2 protein, it would be more reliable to validate the knockout strategy used for this figure with a PCR or a WB. The same comment applies to Figure 3.
Figure 5H: Why aren't the results with dGgnbp2 OE shown in the locomotion sets, but rather a het null + GGNBP2OE is reported? This does not match the groups represented in the previous panels of the same figure.
Line 223 typo: In motor neurons, it was localized to the cytoplasm
Figure 6: I suggest performing N/C fractionation and WB probing for V5 to better characterize protein localization. This data is relevant given results in the next figure.
Figure 6I:To claim dGgnbp2 function is cytoplasmic, data on mutant alone needed (not in null background).
Figure 7: The volcano plot shows that the over expression system and the insertion only exhibit similar genes, which suggests that most of the observed changes are due to the insertion alone. As we do not have a clear understanding of GGNBP2's function in the nucleus, we need more precise data on over expression.
Figure 8: Autophagy experiments are very interesting, despite the lack of crucial data. While it is evident that autophagy dysregulation occurs due to dGgnbp2 dysregulation, it is unclear whether it is a direct cause of the pathology. This assumption can only be confirmed by conducting a few experiments. Firstly, auto-Nagy defects should be measured in rescue models such as the one presented in figure 5. Secondly, treatment should be administered to restore autophagic flux, such as rapamycin or Torin1, as these drugs are known to help in cases of autophagy dysregulation. Another experiment that could be conducted is the overexpression of key autophagic proteins, such as Atg8.
Significance
This study is unique as few studies have focused on this protein and none on its role in motor neurons. The experiments were well-conducted, with the proper controls in place. The authors clearly demonstrate the significance of balanced protein levels for proper synapsis development and optimal motor neuron performance. The study also evaluated RNA dysregulation in different models used in the previous section of the paper. The authors found that autophagy was one of the dysregulated pathways. They characterized the autophagic defects in these cells.
The study would be of interest to a specialized audience since its potential translational implications.
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Referee #1
Evidence, reproducibility and clarity
In this paper, Kerwin et al. investigate the role of the GGNBP2 in synaptic morphology and autophagy in motor neurons. Using Drosophila, the authors performed functional studies of GGNBP2, a putative nuclear protein which has been linked to ALS through GWAS. Through creating clean mutants using CRISPR, the authors found that the null mutants of the fly homolog of GGNBP2 (CG2182, a previously uncharacterized gene which they propose to name Ggnbp2) are viable and fertile, but exhibit motor defects in adult flies accompanied by synaptic phenotypes in the larval neuromuscular junction (NMJ). In addition, the authors show that …
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Referee #1
Evidence, reproducibility and clarity
In this paper, Kerwin et al. investigate the role of the GGNBP2 in synaptic morphology and autophagy in motor neurons. Using Drosophila, the authors performed functional studies of GGNBP2, a putative nuclear protein which has been linked to ALS through GWAS. Through creating clean mutants using CRISPR, the authors found that the null mutants of the fly homolog of GGNBP2 (CG2182, a previously uncharacterized gene which they propose to name Ggnbp2) are viable and fertile, but exhibit motor defects in adult flies accompanied by synaptic phenotypes in the larval neuromuscular junction (NMJ). In addition, the authors show that overexpression of Ggnbp2 also cause behavioral and NMJ defects, which is significant for ALS studies since the variant associated with this condition seems to increase the levels of GGNBP2 based on eQTL studies. Interestingly, the human GGNBP2 was able to rescue the fly LOF mutant phenotypes, suggesting that they have conserved molecular functions. Surprisingly, while mammalian GGNBP2 has been suggested to function as a transcription factor and gain and loss of this gene seems to mildly alter the transcriptome in flies, the authors showed that majority of the endogenously expressed fly Ggnbp2 protein is found in the cytoplasm and that the predicted nuclear localization signal (NLS) is not required for its function in motor neurons. Finally, the authors performed some additional experiments to propose a functional link between this gene and autophagy, focusing on its potential regulation of PI(3)P and genetic interaction with a fly ortholog of TBK1, which have also been linked to ALS in human.
Overall, I feel this work addresses an important question in the field and the genetics experiments have been conducted with rigor. This study somewhat lacks mechanistic insights (e.g. how does Ggnbp2 regulate PI(3)P and motor neuron function?) but there are a number of novel findings (e.g. first generation and characterization of the null mutant of Ggnbp2 in flies, showing that it's predicted NLS is not important) that makes this paper provide value to the literature and community in its current form. While I have several major and minor issues that I would like to see addressed the authors, I would be generally in favor of this paper to be published in an appropriate journal that targets readers with interests in human neurological disorders and Drosophila biology.
Major Points
Major #1: In Figs 5, 6 and S3, the authors demonstrated significant rescue of Ggnbp2 null phenotypes by overexpressing fly Ggnbp2 or human GGNBP2 protein using the GAL4/UAS system. However, data shown in Fig 3 and elsewhere reveals that overexpression of fly Ggnbp2 results in smaller bouton numbers and larger boutons. Regarding this...
#1A: Does overexpression of human GGNBP2 in a wild-type background show similar NMJ and motor behavioral defects as fly Ggnbp2?
#1B: It is quite surprising that the authors were able to rescue the null mutant NMJ phenotype using GAL4/UAS (in this case OK6-GAL4) system considering that overexpression of this protein seems to have a strong effect using this driver as well. Is this because they used the UAS-dGgpnb2::V5 as a heterozygous in FigS3, which is a condition in which the overexpression phenotype is not seen? If so, the genotype of FigS3 (and Fig3) should be matched with FigS4 (otherwise, it looks like the authors used homozygous of the UAS in FigS3).
Major #2: To assess adult fly locomotor performance, the authors employed the negative geotaxis assay to measure their climbing activity (Fig4). While the data show that the flies with LOF or GOF of Ggnbp2 have age-dependent defects, it is possible that the effect is developmental, especially for the overexpression paradigm. Considering that ALS is considered to be an adult onset neurodegenerative disease, it would be valuable if the authors can perform a conditional overexpression study of Ggnbp2[OE-EPgy2] using the Gal80[ts] system in which the fly Ggnbp2 that is overexpressed post-developmentally (i.e. overexpression of this protein induced only after eclosion) can also have an age-dependent motor defect. Considering that the authors do not perform any synaptic studies in adults (i.e. all NMJ experiments are performed in the larva), such experiments will increase the value of this work in the context of ALS research.
Major #3: The authors generated several of UAS-fly Ggnbp2 (V5 tagged with or without the NLS) and UAS-human GGNBP2 (Myc tagged). Regarding these...
#3A: Other than in Fig6A, the authors do not show their expression pattern in motor neurons. It would appreciate if the authors can provide an immunostaining image of the all three proteins in the cell body and neurons of flies when expressed using OK6 or OK371. This way, the readers can appreciate whether the human and fly proteins behave similarly, and whether the deletion of the predicted NLS alters the subcellular localization of the protein. I acknowledge that it may be already difficult to observe the wild-type Ggnbp2 in the nucleus so one may not see a major difference but it would be important to document these.
#3B: Considering that the Ggnbp2::V5 seems to show a punctate pattern, may be interesting to see if this signal overlaps with the Atg8a, PIP2 and 2xFYVE::GFP in the cell body or in the synapse.
Minor points
Minor #1: In line 62, "Given that 75% of genes..." needs a minor correction, as 77% is the the number of gene that is mentioned in the cited reference (77%). Perhaps the authors can say "Given that about 75% of genes...".
Minor #2: In line 266, the title of this section is "dGgnbp2 is linked to autophagy in motor neurons" but the author only shows data regarding the genetic interaction between Ggnbp2 and ik2 (official gene name is IKKε in FlyBase) in this section. Although IKKε and its mammalian homolog TBK1 is known to regulate autophagy, these are kinases that are involved in other processes (e.g. cell proliferation, cell death, cell polarity) so the title is a bit of an overstatement. Since the connection to autophagy is more directly shown in subsequent sections, I would recommend modifying the title of this section (e.g. "dGgnbp2 genetically interacts with IKKε, an ortholog of mammalian TBK1"". Also, note that IKKε is orthologous to both TBK1 and IKBKE so this may need to be clearly mentioned in the text.
Minor #3: In line 282, the loss-of-function (lof) allele for ik2[1] requires proper reference that experimentally showed that this is indeed a lof allele. Also, please change the 'ik2' nomenclature to 'IKKε' to match with the latest official gene name.
Minor #4: In FigS2, can the author show where the predicted NLS of the fly protein is that they deleted in Fig6E so the readers can see how conserved this region is between the fly and human proteins?
Minor #5: I personally feel that the section regarding "RNA-seq analysis of Ggnbp2" is a bit out of place. Currently, this follows the section that says Ggnbp2 is does not function in the nucleus, so it doesn't make much sense to perform RNA-seq experiment for something that you think primarily works in the cytoplasm if the goal of this assay was to find direct mechanistic targets. Perhaps the authors can consider showing moving this section to before the "dGgnbp2 functions in the cytoplasm of motor neurons" (and place Fig7 before Fig6) so you can use the fact that you didn't see much dramatic gene expression changes in the LOF/GOF mutants as a rationale of why you decided to question its nuclear requirements. Just a suggestion, but this may make your paper flow better.
Significance
Overall, I feel this work addresses an important question in the field and the genetics experiments have been conducted with rigor. This study somewhat lacks mechanistic insights (e.g. how does Ggnbp2 regulate PI(3)P and motor neuron function?) but there are a number of novel findings (e.g. first generation and characterization of the null mutant of Ggnbp2 in flies, showing that it's predicted NLS is not important) that makes this paper provide value to the literature and community in its current form. While I have several major and minor issues that I would like to see addressed the authors, I would be generally in favor of this paper to be published in an appropriate journal that targets readers with interests in human neurological disorders and Drosophila biology.
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