Axon guidance genes modulate neurotoxicity of ALS-associated UBQLN2

  1. Sang Hwa Kim
  2. Kye D Nichols
  3. Eric N Anderson
  4. Yining Liu
  5. Nandini Ramesh
  6. Weiyan Jia
  7. Connor J Kuerbis
  8. Mark Scalf
  9. Lloyd M Smith
  10. Udai Bhan Pandey
  11. Randal S Tibbetts

  • Curated by eLife

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    eLife assessment

    This valuable study carried out a genetic screening of Drosophila lines expressing wild-type or ALS/FTD mutations of ubiquilin 2, and identified several suppressors and enhancers of ubiquilin 2 phenotypes. The study particularly focused on two genes involved in axon guidance pathways, unc5 and beat-1b. The evidence supporting the conclusions is solid, although some of the presented data are unrelated to the main findings, which detracts from the focus of the work. This work will be of interest to a broad audience studying ALS/FTD and neurodegenerative diseases.

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Abstract

Mutations in the ubiquitin (Ub) chaperone Ubiquilin 2 (UBQLN2 ) cause X-linked forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) through unknown mechanisms. Here, we show that aggregation-prone, ALS-associated mutants of UBQLN2 (UBQLN2 ALS ) trigger heat stress-dependent neurodegeneration in Drosophila . A genetic modifier screen implicated endolysosomal and axon guidance genes, including the netrin receptor, Unc-5, as key modulators of UBQLN2 toxicity. Reduced gene dosage of Unc-5 or its coreceptor Dcc/frazzled diminished neurodegenerative phenotypes, including motor dysfunction, neuromuscular junction defects, and shortened lifespan, in flies expressing UBQLN2 ALS alleles. Induced pluripotent stem cells (iPSCs) harboring UBQLN2 ALS knockin mutations exhibited lysosomal defects while inducible motor neurons (iMNs) expressing UBQLN2 ALS alleles exhibited cytosolic UBQLN2 inclusions, reduced neurite complexity, and growth cone defects that were partially reversed by silencing of UNC5B and DCC . The combined findings suggest that altered growth cone dynamics are a conserved pathomechanism in UBQLN2-associated ALS/FTD.

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  1. Version published to 10.7554/elife.84382 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    1. The heat shock effect in the drosophila lines was not understood in the study. Why did some lines show phenotypes only at 29C but not 22C? The study showed data that ubiquilin 2 expression was not impacted by 29C, then what caused the phenotypic differences? In addition, the method section did not describe clearly whether a temperature sensitive promoter was used in the flies.

    The heat inducibility of the UBQLN2 transgenes is likely attributed to heat shock elements in the UAS promoter as noted in on page 6, line 4-14. The heat inducibility of dUbqln is interesting and may reflect transcriptional and/or posttranscriptional mechanisms. While it is possible that increased UBQLN2 contributes to the severe phenotypes in UBQLN24XALS flies reared at 29C; this is not seen for UBQLN2WT and UBQLN2P497H flies. Instead, we postulate that heat stress synergizes with the misfolded UBQLN24XALS protein to disrupt proteostasis and/or endolysosomal function. This clarification has been added to paragraph 2 of the Discussion (page 16, line 15-25) section of the revised MS: “The reason for enhanced toxicity of UBQLN24XALS is unclear; however, its enhanced aggregation potential may overwhelm cellular proteostasis machinery and/or accelerate disease mechanisms that are slow to manifest in neurons harboring ALS point mutations. This is consistent with the fact that UBQLN24XALS toxicity in flies was unmasked by HS, which is a well-known inducer of proteotoxicity.” We have also explicitly state the HS inducibility of the UAS-Gal4 in the revised Materials and methods (page 20, line 24-25).

    1. The study showed data on male and female flies separately in some but not all experiments. In addition, the manuscript largely avoided discussing whether there was a sex difference in those experiments.

    We showed separate male and female eye phenotypes in Figure 1 to clearly demonstrate that UBQLN24XALS toxicity is not sex dependent. Subtle sex differences were seen in the longevity and climbing assays and were reported in figures 4A and 4D. In Figure 4D, Unc-5 silencing extended the lifespan of Elav>Gal4 female control flies but not Elav>Gal4 male control flies. In Figure 4A, an Unc-5 KK RNAi line rescued climbing of D42>UBQLN24XALS male flies, but not female flies (a second Unc-5 RNAi line rescued both males and females). The reasons for sex differences in these specific experiments is unclear.

    1. Some data appear to be peripheral with no significant contribution to the main findings. Moreover, some data were introduced but were not explained. For instance, the RNA-Seq analysis (Fig 2) did not contribute much to the study. The rescue effect of UBA* (F594A mutant) in Fig 1-Supplemental 1B was interesting but was not elaborated or followed up. FUS flies in Fig 6-Supplement 2 were abrupted introduced with little discussion.

    We understand the reviewer’s point or the reviewer’s point is well taken. Appreciating the reviewer’s comment, we moved both figures to the supplementary data.

    RNA-Seq (Fig. 2)

    Although not essential, the RNA-Seq adds experimental rigor to the study by providing strong molecular correlates to eye degeneration phenotypes across different UBQLN2 genotypes. It shows the unique toxicity of UBQLN24XALS and reinforces phenotypic similarity between UBQLN2WT and UBQLN2P497H flies, which likely reflects non-specific toxicity of overexpressed UBQLN2 proteins. We have carried out additional data analyses requested by the reviewer and moved the RNA-Seq data to Figure 1-figure supplement 2.

    UBA mutant (Figure1-figure supplement 1)

    Both aggregation and toxicity of UBQLN24XALS were abolished by an inactivating F594A mutation in the UBA domain. While this implicates Ub binding in the biochemical mechanism of UBQLN2 toxicity, we have not followed up on the finding in either fly or iMN models and have chosen to remove the data (Figure1-figure supplement 1) from the revised MS.

    Lack of genetic interaction between FUS and Unc-5 (Figure 3-figure supplement 1).

    This data was included to show that shUnc-5 is not a general suppressor of eye toxicity in Drosophila. This contrasts with lilliputian, whose mutation rescues toxicity phenotypes elicited by FUS, TDP-43, and UBQLN2. We believe that the FUS control data enhances experimental rigor and have retained the data in the revised MS, with some additional clarification on page 10, line 5-8.

    1. The main quadrupole (4XALS) mutation used in the study was not found in patients. The relevance of the findings needs to be thoroughly justified.

    The use of combinatorial mutants—either in the same gene or same pathway—can sometimes be used to enhance neurodegenerative phenotypes in cellular and rodent models for neurodegenerative diseases, most notably, Alzheimer’s Disease. In the case of the 4XALS mutant, we reasoned that its enhanced aggregation might drive stronger phenotypes than those elicited by UBQLN2 clinical alleles, whose toxicity is barely discernible in flies (relative to overexpressed UBQLN2WT) or in iMNs. We have clarified the rationale for testing the 4XALS mutant and articulated its potential strengths and weaknesses in Results (page 5, line 14-page 6, line 2) and Discussion (page 16, line 15-25) sections.

    1. ALS and FTD are age-related neurodegenerative diseases, whereas the involvement of axon guidance genes in indicative of disruptions during the developmental stage. The manuscript did not discuss this potential caveat.

    We have inserted the following sentence in the discussion to note this caveat: “Consistent with this notion, UNC5B has been linked to neurodegeneration in the 6-OHDA model of Parkinson’s Disease (PD) and UNC5C has been nominated as a risk allele in late-onset Alzheimer’s Disease. Defining the contributions of pathologic UNC5 signaling to the development or progression of ALS-dementia awaits further study.” on Page 20, line 2-6. We have added a similar sentence to the Limitations paragraph at the end of the Discussion: “Third, it is possible that axon guidance genes are only relevant to UBQLN2 toxicity in the context of the developing nervous system”.

  3. eLife

    eLife assessment

    This valuable study carried out a genetic screening of Drosophila lines expressing wild-type or ALS/FTD mutations of ubiquilin 2, and identified several suppressors and enhancers of ubiquilin 2 phenotypes. The study particularly focused on two genes involved in axon guidance pathways, unc5 and beat-1b. The evidence supporting the conclusions is solid, although some of the presented data are unrelated to the main findings, which detracts from the focus of the work. This work will be of interest to a broad audience studying ALS/FTD and neurodegenerative diseases.

  4. eLife

    Reviewer #1 (Public Review):

    Kim et al carried out a genetic screening using Drosophila lines to identify genetic modifiers of ubiquilin 2 mutations associated with ALS/FTD. They generated Drosophila lines expressing wild-type or various mutations of ubiquilin 2 and used the rough eye phenotype as the primary screening criterion. They used the deficiency library in the screening and subsequently attempted to narrow down to single genes. They identified multiple suppressors and enhancers from the deficiency lines and carried out further studies on an endosomal gene rab5, an axon guidance gene unc5 and its co-receptor frazzled, and another axon guidance gene beat-1b. Critical findings were also confirmed in iPSC and induced motor neurons (iMNs), supporting the relevance of the findings in human neurons. The study is important as it provides compelling evidence linking axon guidance/synaptic maintenance to ubiquilin 2-mediated neurotoxicity.

    With the above strengths and impact, there are several weaknesses. First, the heat shock effect in the drosophila lines was not understood in the study. Why did some lines show phenotypes only at 29C but not 22C? The study showed data that ubiquilin 2 expression was not impacted by 29C, then what caused the phenotypic differences? In addition, the method section did not describe clearly whether a temperature sensitive promoter was used in the flies. Second, the study showed data on male and female flies separately in some but not all experiments. In addition, the manuscript largely avoided discussing whether there was a sex difference in those experiments. Third, some data appear to be peripheral with no significant contribution to the main findings. Moreover, some data were introduced but were not explained. For instance, the RNA-Seq analysis (Fig 2) did not contribute much to the study. The rescue effect of UBA* (F594A mutant) in Fig 1-Supplemental 1B was interesting but was not elaborated or followed up. FUS flies in Fig 6-Supplement 2 were abrupted introduced with little discussion. Fourth, the main quadrupole (4xALS) mutation used in the study was not found in patients. The relevance of the findings needs to be thoroughly justified. Lastly, ALS and FTD are age-related neurodegenerative diseases, whereas the involvement of axon guidance genes in indicative of disruptions during the developmental stage. The manuscript did not discuss this potential caveat.

    Overall, this study identified several potential genetic modifiers of ubiquilin 2 in the context of ALS/FTD. It represents a significant advancement of our understanding of ubiquilin 2-mediated ALS/FTD and related neurodegenerative diseases.

  5. eLife

    Reviewer #2 (Public Review):

    In the present article, the author aimed at finding disease-modifier for a disease that still nowadays is incurable. To do so the authors decided to employ a drosophila model of ALS, bearing four mutations on the Ubiquilin gene. The model displays eye and motoneuron phenotypes serving as a valuable platform for genetic screenings. The screening performed in the present work shows many suppressors and enhancers of the toxicity associated with the presence of the 4 Ubiquilin mutations. The authors then strengthen the findings of the screening by validating some hits and by studying more in details one involved in the axon guidance signaling. They found that suppressing Unc5 and DCC leads to a less severe phenotype in the flies. They then suppress the ligand of the Unc5 receptor and found that also this approach relieves the phenotype. They then confirmed this results in iPSCs by creating a new cell line harboring the four mutations. They found that the neurites defects found in the mutated UBQLN iPSC was rescued by suppressing Unc5 and DCC. This study has relevance to the ALS field as many of the findings can be harnessed to develop drugs suited for ALS patient bearing Ubiquilin mutations. I think that the major weaknesses of this paper are (i) the fact that they focus on just one mutation, which is pretty rare, while probably most of findings should be also validated in models of sporadic ALS (iPSCs lines). (ii) The amount of data presented, for as much as it is technically well-performed, does not help the reader to focus the attention of the main point which is Unc5 signaling relevance in Ubiquilin associated ALS.

  6. Version published to 10.1101/2022.10.31.514355v1 on bioRxiv