A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency

MBoC ARTICLE A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency Abigail Cline a, *, Ningguo Gao b, *, Heather Flanagan-Steet
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MBoC ARTICLE A zebrafish model of PMM2-CDG reveals altered neurogenesis and a substrate-accumulation mechanism for N-linked glycosylation deficiency Abigail Cline a, *, Ningguo Gao b, *, Heather Flanagan-Steet a, Vandana Sharma c, Sabrina Rosa d, Roberto Sonon a, Parastoo Azadi a, Kirsten C. Sadler d, Hudson H. Freeze c, Mark A. Lehrman b, and Richard Steet a a Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602; b Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; c Sanford Children s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; d Division of Liver Diseases/Department of Medicine, Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY ABSTRACT Congenital disorder of glycosylation (PMM2-CDG) results from mutations in pmm2, which encodes the phosphomannomutase (Pmm) that converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P). Patients have wide-spectrum clinical abnormalities associated with impaired protein N-glycosylation. Although it has been widely proposed that Pmm2 deficiency depletes M1P, a precursor of GDP-mannose, and consequently suppresses lipid-linked oligosaccharide (LLO) levels needed for N-glycosylation, these deficiencies have not been demonstrated in patients or any animal model. Here we report a morpholino-based PMM2-CDG model in zebrafish. Morphant embryos had developmental abnormalities consistent with PMM2-CDG patients, including craniofacial defects and impaired motility associated with altered motor neurogenesis within the spinal cord. Significantly, global N-linked glycosylation and LLO levels were reduced in pmm2 morphants. Although M1P and GDP-mannose were below reliable detection/quantification limits, Pmm2 depletion unexpectedly caused accumulation of M6P, shown earlier to promote LLO cleavage in vitro. In pmm2 morphants, the free glycan by-products of LLO cleavage increased nearly twofold. Suppression of the M6P-synthesizing enzyme mannose phosphate isomerase within the pmm2 background normalized M6P levels and certain aspects of the craniofacial phenotype and abrogated pmm2- dependent LLO cleavage. In summary, we report the first zebrafish model of PMM2-CDG and uncover novel cellular insights not possible with other systems, including an M6P accumulation mechanism for underglycosylation. Monitoring Editor Reid Gilmore University of Massachusetts Received: Jun 5, 2012 Revised: Aug 10, 2012 Accepted: Aug 29, 2012 This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/ /mbc.e ) on September 5, *Joint lead authors. Address correspondence to: Mark A. Lehrman Richard Steet Abbreviations used: CDG, congenital disorder of glycosylation; CH, ceratohyal; dpf, day postfertilization; Dol, dolichol; ER, endoplasmic reticulum; ERAD, ERassociated degradation; FACE, fluorophore-assisted carbohydrate electrophoresis; F6P, fructose-6-phosphate; G 3 M 9 Gn 2 -P-P-Dol, glucose 3 mannose 9 N-acetylglucosamine 2 -pyrophosphate-dolichol; hpf, hour postfertilization; LLO, lipid-linked oligosaccharide; M1P, mannose-1-phosphate; M6P, mannose-6-phosphate; MO, morpholino; Pmm2, phosphomannomutase 2; PQ, palatoquadrate; SB, splice blocker; TB, translation blocker Cline et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution Noncommercial Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). ASCB, The American Society for Cell Biology, and Molecular Biology of the Cell are registered trademarks of The American Society of Cell Biology. INTRODUCTION The congenital disorders of glycosylation (CDGs) are a group of heterogeneous, hypomorphic, inherited diseases characterized by deficient N-glycosylation, typically detected by underglycosylation of serum glycoproteins (Jaeken and Matthijs, 2001; Marquardt and Freeze, 2001). Traditionally divided into two groups, the type I CDGs are classified by genetic defects at loci known to participate in the biosynthesis of the lipid-linked oligosaccharide (LLO) glucose 3 mannose 9 N-acetylglucosamine 2 -pyrophosphate-dolichol (G 3 M 9 Gn 2 -P- P-Dol), the glycan donor for N-glycosylation. This includes loci encoding proteins and enzymes involved in sugar and lipid metabolism, endoplasmic reticulum (ER)-localized glycosyltransferases, and proteins that facilitate utilization of substrates (Haeuptle and Hennet, 2009). First recognized in the clinic by Jaeken in the 1980s, the most common subtype of CDG is caused by deficiency of the metabolic Volume 23 November 1, enzyme phosphomannomutase 2 (Pmm2; Matthijs et al., 1997). This enzyme converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P), the substrate needed for the synthesis of GDP-mannose and the eventual production of LLO. Designated PMM2-CDG (this subtype was formerly referred to as CDG-Ia), patients with this disease exhibit a constellation of abnormalities, including psychomotor and mental retardation, cerebellar atrophy, peripheral neuropathy, hypotonia, and ataxia (de Lonlay et al., 2001; Coman et al., 2008; Freeze et al., 2012). There are no current therapies for PMM2- CDG, and little is known about the relationship between the underglycosylation of proteins and the clinical features of the disease (Freeze, 2001, 2009). Our limited understanding of type I CDG pathophysiology can be attributed to the paucity of suitable animal models for these diseases. Although many of the predicted CDG biochemical phenotypes have been confirmed by analyses of LLO and glycoprotein synthesis in cultured fibroblasts and leukocytes from patients, such culture systems fail to recapitulate the more complex physiological relationships that exist within the entire organism, yielding inconsistent results (Gao et al., 2005). These limitations have hampered evaluation of potential and actual therapeutic treatments for type I CDGs. For example, dietary mannose supplementation, which is expected to increase intracellular M6P concentrations and drive M1P synthesis by mass action, is clinically beneficial for MPI-CDG (also known as CDG- Ib) patients but not for PMM2-CDG patients. A mouse line completely lacking mannose phosphoisomerase (MPI), the enzyme responsible for the generation of M6P from fructose 6-phosphate (F6P), is an embryonic lethal (DeRossi et al., 2006). Attempts to generate viable mouse models for PMM2-CDG, the most common form of CDG, have also been particularly challenging. Complete knockout of Pmm2, or knock-in of the most common human PMM2-CDG allele, R137H, into this null background resulted in early embryonic lethality (Thiel et al., 2006). In contrast, knock-in of another common allele, F118L, resulted in only mild loss of enzymatic activity and no detectable phenotypes (Schneider et al., 2012). Compound heterozygotes with both the R137H and F118L alleles, however, survived to embryonic day 9.5 (Schneider et al., 2012). These embryos had reduced staining with wheat germ agglutinin, a plant lectin able to bind to sialic acid and N-acetylglucosamine residues and therefore capable of detecting several classes of glycoconjugates, including N-linked glycans. Strikingly, when the water given to the pregnant dams was supplemented with mannose, many of these embryos were born live, survived past weaning, and regained normal wheat germ agglutinin staining patterns. These data suggest that the R137H/F118L embryos had glycosylation deficiencies that were reversible by maternal mannose supplementation. Despite these findings, a tractable animal model that closely replicates the genetic, developmental, and glycosylation deficiencies documented for PMM2-CDG patients is still needed for a thorough biochemical and behavioral evaluation of the disease mechanisms and experimental treatments. To overcome the present limitations of PMM2-CDG mouse models, we took advantage of the genetic and experimental attributes of zebrafish (Danio rerio), an excellent vertebrate system for both the study of embryonic development and the modeling of genetic diseases. We recently described a model of MPI-CDG (Chu et al., 2012), demonstrating the utility of this system for modeling CDG. Here we report that suppression of pmm2 expression using a morpholino-based approach resulted in morphant embryos that exhibit several cellular and molecular phenotypes consistent with the human disease, including altered craniofacial cartilage development, impaired motility, and reduced protein N-glycosylation. We also provide direct evidence for reduced LLO levels in the morphants. Surprisingly, although our analytical methods were too insensitive to reliably detect M1P and GDP-mannose in zebrafish, an increase in the level of the Pmm2 substrate M6P was consistently observed. Additional genetic and biochemical experiments revealed that loss of N-glycosylation in the pmm2 morphants was best explained by accelerated hydrolysis of LLO triggered by the accumulated M6P. Taken together, these results yield new insights into the cellular and molecular basis of type I CDGs, lend support to the concept of fluxbased therapy, and further highlight the utility of the zebrafish system in the investigation of inherited metabolic disorders. RESULTS Pmm2 activity is shown to be significantly reduced in zebrafish embryos by using a morpholino-based strategy To inhibit pmm2 expression, we tested two different antisense morpholinos (MOs) directed against zebrafish pmm2 one that inhibits mrna translation (translation blocker [TB MO]) and one that inhibits mrna splicing (splice blocker [SB MO]; Figure 1A). Over a μm concentration range, the degree of knockdown with each morpholino was assessed by either reverse transcription (RT)-PCR (SB MO) or Pmm2 activity assays (SB and TB MOs). The SB MO caused a linear decrease in the amount of normally spliced pmm2 transcripts, with a maximal reduction at 0.52 μm (Figure 1B). Although a decrease in normally spliced transcript is often accompanied by a concomitant increase in an alternate splice product (either larger or smaller, depending on the junction targeted), we were unable to detect the larger intron-containing transcript after pmm2 inhibition. To assess whether the lack of this product after PCR amplification was due to the large amplicon size, we probed its presence with two additional sets of primers (see Materials and Methods for details) whose efficacies were previously validated using genomic DNA. In light of the fact that neither primer set amplified the alternate splice product, we conclude that this SB MO caused rapid elimination of the full-length pmm2 mrna. Surprisingly and for reasons that are unclear, pmm2 transcript abundance increased with higher MO concentration (Figure 1B). To determine the effect of MO inhibition on protein expression, we measured Pmm2 activity in lysates using a coupled assay. Pmm2 activity was reduced to 33.4% of control with the SB MO, which paralleled the RT-PCR results, and to 58% of control with the TB MO. However, like the corresponding message, the enzymatic activity of Pmm2 could not be completely suppressed, and it rebounded at the highest SB MO concentration, suggesting the presence of compensatory mechanisms (Figure 1C). Based on these considerations, 0.52 μm SB MO was used for the remainder of the study. At this concentration, Pmm2 activity was stably reduced for the first 4 d of development (unpublished data). Coinjection of the SB MO and full-length pmm2 mrna (whose sequence is nonhomologous to the SB MO) rescued Pmm2 activity to near-normal levels, demonstrating both the specificity of the SB MO and the efficacy of the injected mrna (Figure 1D). In situ hybridization analyses of pmm2 revealed global expression throughout the heads of control embryos, with transcript concentration notably increased in ventral regions (Figure 1E; arrows highlight ventral concentration). Low levels of pmm2 expression were also noted in embryonic spinal cords (unpublished data). Consistent with decreased enzyme activity, both transcript patterns were clearly reduced in embryos injected with the pmm2 SB MO (Figure 1E). pmm2 morphants exhibit alterations in craniofacial cartilage development Motivated by the fact that PMM2-CDG patients exhibit craniofacial and skeletal malformations, we were prompted to analyze 4176 A. Cline et al. Molecular Biology of the Cell FIGURE 1: Injection of pmm2 directed antisense morpholinos into zebrafish embryos reduces pmm2 transcript abundance and activity. (A) Schematic representation of pmm2 gene. The positions of TB and SB MOs are indicated, as is the transcriptional start site (*). Arrows indicate the position of one set of primers used to assess transcript abundance (see Materials and Methods). (B) RT-PCR of 3-dpf embryos after injection of indicated concentrations of SB MO. (C) Pmm2 activity measurements of 3-dpf embryos after injection of either the TB or SB MO. n = 4 experiments. Throughout this article, *p 0.05 and **p 0.01 (Student s t test). (D) Measurement of Pmm2 and Mpi activity in 3-dpf control embryos, embryos injected with 0.52 μm SB, and mrna-rescued morphants. n = 25 experiments. (E) In situ analysis of pmm2 expression in control and pmm2 morphant (MO) embryos. Arrowheads indicate ventral concentration of staining. developing cartilage in pmm2 morphants in detail by staining with Alcian blue, a dye that binds acidic components of the extracellular matrix. pmm2 morphant embryos displayed several defects in cartilage morphogenesis, including protracted Meckel s (M) cartilages, misshapen palatoquadrate (PQ) and ceratohyal (CH) structures, and kinked pectoral fins (Figure 2A). Each of these phenotypes was rescued by coinjection of pmm2 mrna, suggesting that they were specific to Pmm2 reduction and not potential offtarget MO effects. Furthermore, simultaneous suppression of pmm2 and p53 did not ameliorate the craniofacial phenotypes (unpublished data), indicating that the pmm2 MO does not induce these abnormalities by promoting nonspecific apoptosis. This is an important consideration, as several studies have shown that one common off-target effect of morpholinos is the induction of p53- directed apoptosis (Ekker, 2000; Bill et al., 2009). Measurements of the cartilage structures showed that they were not only misshapen, but also shorter than control cartilages (Figure 2B). In addition, the distance between the M and CH cartilages was reduced by 35% in the pmm2 morphants. With the exception of the length of the CH cartilage, coinjection of pmm2 mrna significantly improved the size and shape of the craniofacial cartilages (Figure 2B). The lack of CH recovery and partial PQ recovery may be accounted for by the fact that these structures finish developing after the M cartilage and may therefore be outside the window of mrna rescue. Although analyses of control and morphant flat-mounted cartilages showed no significant differences in the total number of cells (unpublished data), they revealed alterations in the morphology of morphant chondrocytes (Figure 2C). Unlike control chondrocytes, which were oblong in shape and had converged to form a single line of cells, morphant cells remained round and were arranged in multicellular layers, yielding a cobblestone -like appearance. To quantitatively measure these differences in cellular shape, we calculated the ratio between the short and long axes of individual chondrocytes. The ratio between these axes was significantly larger in the morphants (Figure 2D), reflecting increased cellular roundness. Again, coinjection of pmm2 mrna restored the typical oblong cellular morphology of the morphant chondrocytes. To ask whether the alterations in cell shape reflected an early defect in chondrocyte maturation, we stained control and morphant embryos with peanut agglutinin (PNA), which labels prechondrocytic mesenchymal cells, and type II collagen, one of the earliest markers of chondrocyte differentiation. PNA is a lectin that binds the terminal disaccharide Gal-β(1-3)-GalNAc in O-linked glycans, and is therefore not directly dependent on the mannose-derived products of Pmm2. No differences in the intensity, distribution, or timing of the expression of either marker were detected between control and morphant chondrocytes, suggesting that initial aspects of differentiation are unaffected by pmm2 knockdown (unpublished data). Collectively these findings indicate that reduced Pmm2 activity is associated with changes in craniofacial cartilage formation resulting from altered chondrocyte morphogenesis. pmm2 morphants exhibit multiple motility defects Because PMM2-CDG patients exhibit hypotonia, ataxia, and delayed motor development, we investigated whether pmm2 morphants demonstrated motor system defects. These analyses revealed multiple deficits at two developmental stages in morphant embryos when compared with controls. By 1 d postfertilization (dpf), normal zebrafish embryos exhibit two different motility behaviors: spontaneous movements, consisting of slow, alternating tail flexures, and elicited movements, in which embryos respond to external stimuli with several rapid tail flexures. Analyses of 1-dpf embryos revealed a slight but significant decrease in the number of spontaneous tail curls generated by pmm2 morphants (11.0 vs. 8.2; Figure 3A). In addition, pmm2 morphants exhibited reduced response to touch at 1 dpf. Although both produced a similar number of tail Volume 23 November 1, 2012 Zebrafish model for PMM2-CDG 4177 FIGURE 2: pmm2 morphants have dysmorphic craniofacial cartilage. (A) Alcian blue stains of 4-dpf control and pmm2 morphant embryos revealed altered size and shape of Meckel s cartilage, as well as the palatoquadrate and ceratohyal cartilages. These defects were rescued by coinjection of pmm2 mrna. n = embryos per condition over three experiments. Arrow shows kinked pectoral fins in the pmm2 morphants. (B) The length of individual structures was measured and normalized to a standard length (SL), which was set as the distance between the eyes. Individual cartilage measurements are outlined in the fish schematic. n = embryos per condition over three experiments. Here and throughout this article, ***p (C) The 4-dpf WT, pmm2 morphant (MO), and pmm2 mrna rescued embryos were dissected and the cartilages mounted flat. Analysis of these preparations showed that morphant chondrocytes were rounder and more underintercalated compared with WT chondrocytes. This was rescued by coinjection of pmm2 mrna. n = embryos per condition in three experiments. (D) Chondrocyte shape was measured in each of the affected structures by determining the ratio of cell short axis to long axis. The closer this number is to 1, the rounder is the cell. n = embryos per condition in three experiments. curls following stimulation, 88% of control embryos responded with a complete curl that made contact with the body. In contrast, the majority of morphant embryos (75%) only flexed the tip of their tail, never touching the body. By 3 dpf, deficits in touch responsiveness within the pmm2 morphants were further illustrated by both aberrant escape and swimming behaviors. To quantify these behaviors, we placed 3-dpf embryos in the center of a plate marked with a series of concentric rings, which were used to assign swimming distances (Figure 3B). After stimulation, control embryos robustly swam away from the stimulus, ultimately crossing multiple rings. In contrast, morphant embryos typically swam in a circle toward the stimulus, remaining within the first ring. Both the early-stage (1 dpf) and late-stage (3 dpf) defects in elicited motility were significantly (in 50% of the embryos assayed) rescued by coinjection of pmm2 mrna (Figure 3C). Taken together, these data sug
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