Molecules of ribonucleic acid (or RNA for short) have many roles in cells, including acting as templates to make proteins. RNA is made of building blocks called nucleotides that are assembled to form strands. The precise order of the nucleotides in an RNA molecule can have a dramatic effect on the role that RNA plays in the body. For example, amyotrophic lateral sclerosis (ALS) is a deadly disease caused by the gradual loss of the nerve cells that control muscle (known as motor neurons). The most common cause of inherited ALS is a genetic mutation that results in some RNA molecules having many more copies of a simple six nucleotide sequence known as GGGGCC than normal cells. RNA molecules with these “GGGGCC repeats” form clumps in motor neurons.

The clumps of RNA molecules also contain proteins, but the identities of these RNA-binding proteins and the roles they play in ALS remain largely unknown. Celona et al. have now identified a new RNA-binding protein called Zfp106, which binds specifically to GGGGCC repeats in mice and fruit flies.

Removing the gene that encodes Zfp106 from mice causes the mice to develop ALS. On the other hand, restoring Zfp106 only to the motor neurons of these mutant mice prevents the mice from developing disease. This suggests that Zfp106’s role is specific to motor neurons. Indeed, fruit flies that have too many copies of GGGGCC develop severe symptoms reminiscent of ALS. Introducing a mammalian version of Zfp106 into these flies prevents them from developing the disease.

The findings of Celona et al. suggest that Zfp106 might be a potential new drug target for treating ALS in humans. The next step following this work will be to find out exactly how Zfp106 regulates normal cellular processes by binding to RNA and how it suppresses ALS-like disease by binding to GGGGCC RNA-repeats.

Expanded GGGGCC repeats in the first intron of the C9orf72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (DeJesus-Hernandez et al., 2011; Renton et al., 2011). The repeat expansion has been identified in more than 40% of familial ALS and in at least 7% of sporadic cases (Majounie et al., 2012). Several gain-of-function mechanisms for how the expanded hexanucleotide repeats cause disease have been proposed. One idea suggests that toxic dipeptide repeat (DPR) proteins generated by repeat associated non-ATG (RAN) translation of the RNA repeats cause neurodegeneration (Kwon et al., 2014; Mizielinska et al., 2014; Mori et al., 2013). Another major proposed gain-of-function mechanism for C9orf72 ALS is via the formation of pathogenic repeat RNA aggregates that sequester one or more RNA binding proteins, leading to altered RNA processing and metabolism in motor neurons (Cooper-Knock et al., 2015; Haeusler et al., 2014; Lee et al., 2013; Prudencio et al., 2015; Sareen et al., 2013).

Zfp106 is a C₂H₂ zinc finger protein with four predicted zinc fingers and seven WD40 domains (Grasberger and Bell, 2005). The mouse Zfp106 gene is located on chromosome 2 at 60.37 cM (Chr2:120,506,820–120,563,843 bp; Genome Reference Consortium, Mouse Build 38). In addition, the human ortholog, ZNF106, is located at human chromosome 15q15.1 (Chr15: 42,412,437–42,491,197; Genome Reference Consortium Human Build 38), a region with strong linkage to a rare recessive familial form of ALS (Hentati et al., 1998), suggesting a possible role in human ALS. Consistent with this notion, mice lacking Zfp106 function show evidence of nondevelopmental neuromuscular degeneration, also suggestive of a possible role for Zfp106 in ALS (Anderson et al., 2016; Joyce et al., 2016; van der Weyden et al., 2011).

Here, we identify a previously unknown role for Zfp106 as an RNA binding protein that specifically interacts with GGGGCC repeats and with a network of ALS-associated RNA-binding proteins, including TDP-43. In addition, we establish that knockout of Zfp106 in mice results in an ALS-like motor phenotype and that transgenic restoration of Zfp106 expression specifically in motor neurons suppresses the neurodegenerative phenotype in knockout mice. Finally, we show that Zfp106 is a potent suppressor of neurotoxicity caused by expression of GGGGCC repeats in a Drosophila model of C9orf72 ALS. Thus, these studies have important implications for the most common form of human ALS.

Sequestration of RNA binding proteins by rGGGGCC repeats has been implicated in the pathology of C9orf72 ALS (Mizielinska and Isaacs, 2014). Therefore, to identify rGGGGCC-binding proteins that may be sequestered by repeat-containing RNA and involved in the pathology of C9orf72 ALS, we performed a pull-down assay using biotinylated r(GGGGCC)₈ RNA and identified the zinc finger protein Zfp106 as a previously unknown rGGGGCC-binding protein. Zfp106 bound specifically to r(GGGGCC)₈ but not the control sequence r(AAAACC)₈ (Figure 1a). RNA electrophoretic mobility shift assay (EMSA) using purified Zfp106 protein demonstrated that Zfp106 bound directly to the rGGGGCC repeats and that this binding was specific since it was efficiently competed by unlabeled self oligonucleotide but not a 30× molar excess of the control oligonucleotide (Figure 1b). Importantly, Zfp106 binding to r(GGGGCC)₄ was not competed by 30× molar excess of a mutated RNA probe of equal length, nor was it competed by 30× molar excess of rCUG repeats comprising a MBNL1 binding site (Figure 1—figure supplement 1a). Likewise, MBNL1, an unrelated RNA-binding protein involved in myotonic dystrophy (Miller et al., 2000), bound efficiently and specifically to rCUG repeats under conditions in which Zfp106 showed no binding to the same rCUG sequence (Figure 1—figure supplement 1b) and, as previously described (Reddy et al., 2013), was not able to bind GGGGCC repeats (Figure 1—figure supplement 1a). Together, these data demonstrate that Zfp106 binds efficiently and specifically to GGGGCC RNA repeats.

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Patients with C9orf72 ALS show evidence of post-mortem nucleolar stress (Haeusler et al., 2014). Interestingly, in cultured Neuro-2a neuronal cells, Zfp106 was primarily present in the nucleolus and in other discrete nuclear puncta (Figure 1c). Likewise, in lower motor neurons from post-mortem human spinal cord samples, ZNF106 was primarily observed in the nucleolus (Figure 1d,d′) and also showed occasional localization in other puncta in the nucleus outside of the nucleolus (Figure 1d′). The localization of Zfp106 to the nucleolus is in accordance with previous studies showing that Zfp106 is predominantly located in the nucleolus of C2C12 myoblast cells (Grasberger and Bell, 2005), and taken together, the results presented in Figure 1 support a possible role for Zfp106 in the biology of C9orf72 neurodegeneration.

As noted above, previous studies have demonstrated a requirement for Zfp106 in neuromuscular function in mice. Abnormal skeletal morphology in Zfp106^{tm1a(KOMP)Wtsi}homozygous mice was briefly reported as part of a large-scale phenotypic screen of genetically modified mice by the Sanger Mouse Genetics Project (van der Weyden et al., 2011). More recently, more extensive phenotypic studies demonstrated progressive neuromuscular disease in the absence of Zfp106 gene function (Anderson et al., 2016; Joyce et al., 2016). These studies, combined with the interaction of Zfp106 with the C9orf72 hexanucleotide repeat sequence, prompted us to examine Zfp106 knockout mice in additional detail and to determine whether the observed defects were autonomous to motor neurons. Therefore, we obtained the previously described Zfp106^{tm1a(KOMP)Wtsi}mice from the KOMP knockout consortium (van der Weyden et al., 2011) and generated the presumptive null allele (Zfp106^lacz; also referred to herein as Zfp106-null or Zfp106^−/−) using a universal germline deleter transgenic mouse line (Ehlers et al., 2014).

Consistent with published studies, we found that loss of Zfp106 function caused a progressive neuromuscular phenotype. Zfp106-null mice were born at normal Mendelian frequency and were indistinguishable from control mice until approximately 4 weeks of age, when 100% of knockout mice began to exhibit evidence of a wasting disease and loss of muscle strength (Figure 2—figure supplement 1; Video 1). Transverse sections of the quadriceps from 4-week-old Zfp106-null mice showed increased variation in fiber size and evidence of grouped atrophy with very few centrally located nuclei (Figure 2a,b), consistent with neurogenic rather than myopathic disease (Baloh et al., 2007). By three months of age, near end stage disease, Zfp106-null mice displayed extensive muscular atrophy and severe degeneration of muscle fibers (Figure 2c,d). The number of choline acetyltransferase (ChAT)-positive motor neurons was also profoundly and significantly reduced in Zfp106 knockout spinal cords compared to littermate control mice (Figure 2e–i), providing further evidence that loss of Zfp106 in mice causes a neurodegenerative phenotype. Independently, we confirmed the knockout phenotype observed with the KOMP allele (van der Weyden et al., 2011) using CRISPR-mediated gene disruption by non-homologous end joining (NHEJ) and creation of a frameshift mutation leading to a premature stop codon. The CRISPR-generated mutation resulted in an essentially identical wasting and histological phenotype (data not shown).

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To gain insight into the cell autonomous requirements for Zfp106, we used the KOMP lacZ knock-in allele (van der Weyden et al., 2011) to examine Zfp106 expression using β-galactosidase activity as a sensitive method for detecting expression. This approach showed Zfp106 expression in both skeletal muscle and motor neurons (Figure 2—figure supplement 2a,b). Given the multiple lines of evidence for a role of Zfp106 in neurodegeneration, and the evident denervation observed in the knockout mice, we hypothesized that the primary requirement for Zfp106 was likely to be in motor neurons. We confirmed a highly significant and dramatic reduction in Zfp106 expression in spinal cord (Figure 2—figure supplement 3a). The ~80% reduction in Zfp106 expression is consistent with previous studies (Anderson et al., 2016; Joyce et al., 2016). Therefore, we used a transgenic approach in mice to restore Zfp106 expression only in motor neurons under the control of the Mnx1 promoter (herein called HB9, HB9-Zfp106) on a Zfp106 germline knockout background (Figure 2j; and Figure 2—figure supplement 3b,c). Importantly, restoration of Zfp106 specifically in motor neurons significantly suppressed the Zfp106 knockout wasting and muscle grip phenotypes and resulted in restoration of the number of ChAT-expressing motor neurons (Figure 2j–m; Figure 2—figure supplement 4), indicating a cell autonomous requirement for Zfp106 in motor neurons.

To gain additional insights into the molecular function of Zfp106, we used mass spectrometry to identify all proteins co-immunoprecipitated by Zfp106 from 293T cells (Figure 3). Several controls were included in the mass spectrometry experiments, including the DNA-binding factors Etv2 and MEF2C and the RNA-binding proteins Vif and Tat. The interactomes for each of the controls were compared to the Zfp106 interactome using the previously described MiST algorithm, which determines specific interactions based on enrichment, reproducibility, and specificity compared to interaction with the negative controls (Jager et al., 2012). These experiments identified thirty-nine proteins that specifically interacted with Zfp106 (Figure 3a; Supplementary file 1). Gene ontology (GO) and protein complex analyses revealed a significant enrichment in interactors with roles in RNA processing, metabolism, and splicing (Figure 3a,b; Supplementary file 2), suggesting that Zfp106 may also play roles in these processes.

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There is growing and compelling genetic and pathological evidence that dysregulated RNA processing and metabolism contribute to the development of ALS and C9orf72 ALS in particular (Ling et al., 2013; Prudencio et al., 2015). Interestingly, several of the Zfp106-interacting proteins identified in our affinity purification-mass spectrometry experiment are proteins directly associated with ALS and/or C9orf72 ALS, including splicing factors TDP-43 and FUS, the nucleolar protein Nucleolin, and the nuclear pore component Nup107 (Freibaum et al., 2015; Haeusler et al., 2014; Lagier-Tourenne et al., 2010; Ling et al., 2013); Figure 3c; Supplementary file 1). We further examined the interaction of Zfp106 with TDP-43 and Nup107, as examples of interacting proteins, by anti-Flag-Zfp106 immunoprecipitation followed by western blot for TDP43 and Nup107 in Neuro-2a cells (Figure 3—figure supplement 1). These experiments confirmed and validated the interaction with these other ALS-associated proteins and support the notion that Zfp106 is part of an RNA metabolic pathway (or pathways) that when dysregulated leads to neurodegeneration.

Prior studies have used Drosophila as an in vivo gain-of-function model of C9orf72 neurodegeneration (Freibaum et al., 2015; Tran et al., 2015; Xu et al., 2013; Zhang et al., 2015). Here, we used the GAL4-UAS system to coexpress mouse Zfp106 and GGGGCC repeats in the fly in a gain-of-function approach to determine if the interaction between Zfp106 and GGGGCC repeats had a functional consequence in this model (Figure 4; Figure 4—figure supplement 1a). Expression of 30 tandem copies of the GGGGCC hexanucleotide in the 5′-untranslated region of a UAS-dependent gfp transcript (30R-GFP) in glutamatergic neurons using OK371-GAL4 (Mahr and Aberle, 2006) caused ~50% lethality due to an apparent failure of flies to eclose from the pupal case (Figure 4a). Furthermore, 28 day-old OK371; 30R-GFP-expressing flies that did manage to successfully eclose exhibited profound defects in locomotor activity compared to control flies (Figure 4b). No overt eclosion or locomotor phenotype was observed in flies expressing GFP only, 3R-GFP, or Zfp106 alone in glutamatergic neurons (Figure 4a,b). Previous studies have also shown that expression of 30R-GFP causes a reduction in the number of active zones in Drosophila larval neuromuscular junctions (Zhang et al., 2015). Consistent with those studies, we found that expression of 30R-GFP reduced active zones as measured by anti-Bruchpilot (Brp) immunofluorescence and quantification (Figure 4c–f). Remarkably, co-expression of Zfp106 with 30R-GFP restored Brp expression at larval active zones (Figure 4c–f) and completely suppressed the ~50% lethality caused by expression of 30R-GFP (Figure 4a). Moreover, co-expression of Zfp106 partially suppressed the adult locomotor defect caused by expression of 30R-GFP (Figure 4b). Importantly, co-expression of Zfp106 did not result in a reduction in the expression of 30R-GFP mRNA Figure 4—figure supplement 1b), indicating that the suppression of the neurotoxic phenotype in Drosophila was not due to dilution of GAL4 protein. Taken together, these data show that Zfp106 is a potent suppressor of neurodegeneration in a C9orf72 ALS model, and the results from this gain-of-function model support the idea that Zfp106 functionally interacts with C9orf72 GGGGCC RNA repeats.

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The ability of Zfp106 to suppress the neurotoxic gain-of-function effect of rGGGGCC repeats has important implications for understanding and possibly ameliorating the pathology of C9orf72 ALS. Previous studies have suggested roles for Zfp106 in rDNA transcription and in pre-rRNA processing (Ide and Dejardin, 2015; Tafforeau et al., 2013). Zfp106 was also identified as a polyadenylated mRNA binding protein in an interactome capture study aimed at defining an atlas of mammalian mRNA binding proteins (Castello et al., 2012). Interestingly, our interactome data also suggest possible roles for Zfp106 in multiple steps of RNA metabolism and processing and interaction with key RNA binding proteins involved in various forms of neurodegeneration. Precisely how disrupting these processes leads to neurodegeneration and exactly how this relates to C9orf72 ALS/FTD remains to be determined. A simple model is that sequestration of Zfp106 by the expanded repeats disrupts normal Zfp106 function in patients with C9orf72 ALS in a sponge-like mechanism (Cooper-Knock et al., 2014; Mizielinska and Isaacs, 2014). Alternatively, one might speculate that Zfp106 plays other functions important in the biology of C9orf72, such as influencing dipeptide repeat protein (DPR) production or affecting nucleolar function or nucleocytoplasmic transport via interactions with Nucleolin or Nup107, which have also been implicated in the pathobiology of C9orf72 ALS (Boeynaems et al., 2016; Freibaum et al., 2015; Haeusler et al., 2014; Jovičić et al., 2015; Kwon et al., 2014; Zhang et al., 2015). Future studies will address the molecular targets and mechanisms of action of Zfp106 in order to gain further insights into the pathogenesis of C9orf72 ALS and to identify new possible therapeutic targets for neuroprotection.

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Author details

1. Barbara Celona

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   BC, Conceptualization, Formal analysis, Validation, Funding acquisition, Investigation, Writing—original draft, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

2. John von Dollen

   Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Contribution

   JvD, Formal analysis, Investigation, Writing—original draft, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

3. Sarat C Vatsavayai

   Department of Neurology, University of California, San Francisco, San Francisco, United States

   Contribution

   SCV, Conceptualization, Formal analysis, Investigation, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

4. Risa Kashima

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   RK, Formal analysis, Investigation, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

5. Jeffrey R Johnson

   Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

   Contribution

   JRJ, Formal analysis, Investigation, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

6. Amy A Tang

   Department of Pathology, University of California, San Francisco, San Francisco, United States

   Contribution

   AAT, Formal analysis, Investigation, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

7. Akiko Hata

   Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States

   Contribution

   AH, Conceptualization, Supervision, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

8. Bruce L Miller

   Department of Neurology, University of California, San Francisco, San Francisco, United States

   Contribution

   BLM, Conceptualization, Funding acquisition, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

9. Eric J Huang

   Department of Pathology, University of California, San Francisco, San Francisco, United States

   Contribution

   EJH, Conceptualization, Formal analysis, Supervision, Writing—review and editing, Final approval of the manuscript

   Competing interests

   The authors declare that no competing interests exist.

10. Nevan J Krogan

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States

    Contribution

    NJK, Conceptualization, Formal analysis, Supervision, Methodology, Writing—original draft, Final approval of the manuscript

    Competing interests

    The authors declare that no competing interests exist.

11. William W Seeley

    1. Department of Neurology, University of California, San Francisco, San Francisco, United States
    2. Department of Pathology, University of California, San Francisco, San Francisco, United States

    Contribution

    WWS, Conceptualization, Funding acquisition, Investigation, Writing—review and editing, Final approval of the manuscript

    Competing interests

    The authors declare that no competing interests exist.

12. Brian L Black

    1. Cardiovascular Research Institute, University of California, San Francisco, San Francisco, United States
    2. Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States

    Contribution

    BLB, Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing, Final approval of the manuscript

    For correspondence

    brian.black@ucsf.edu

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-6664-8913

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