Establishing Zebrafish as a Preclinical Model for Dystroglycanopathies Erin V. Carter, Elisabeth A. Kilroy, Michelle F. Goody, Mary E. Astumian, Joseph J. Belanger, Clarissa A. Henry GSBSE Annual Meeting University of Maine September 16 & 17, 2016

Muscular Dystrophies are Well-Known Genetic Diseases Muscular dystrophies are incurable genetic diseases characterized by muscle wasting and compromised locomotion. Duchenne Muscular Dystrophy is the most common form of the disease, and because of public awareness about DMD, therapeutic strides have been made. However, there is a broad spectrum of muscular dystrophies and potential treatments for these lag behind. It would be ideal to develop treatments that target this large spectrum of conditions. So in order to understand how to tackle this problem, we need to take a look at muscle tissues…

Cell-matrix adhesion complex Muscle Cells Interact With Their Environments Via Cell-Extracellular Matrix Linkages Muscle comes in many shapes and sizes—but regardless of whether we are talking about a human bicep or muscle from a laboratory fish model, muscle is composed of individual fibers linked to one another by cell-extracellular matrix adhesions. The interactions between cell and ECM are called cell-matrix adhesion complexes, and these are critical for muscle fiber viability. A huge amount of muscular dystrophies occur when these linkages are disrupted, including the dystroglycanopathies. When you contract and relax muscle, there is strain on these adhesions, and if they aren’t strong, they will fail… Cell-matrix adhesion complex

Dystroglycanopathies: Phenotypically Diverse Muscular Dystrophies 2° Dystroglycanopathy Genes POMT1 POMT2 POMGnT1 POMGnT2 LARGE FKTN FKRP ISPD DPM1 DPM2 DPM3 DOLK POMK B3GALNT2 B4GAT1 GMPPB TMEM5 …which result from either a lack of dystroglycan or glycosyltransferases that post-translationally modify dystroglycan! Dystroglycan is a protein with two subunits---the beta is linked to dystrophin, and the alpha is heavily glycosylated by a number of enzymes (point to purple box). Presence of both subunits and proper glycosylation is key for laminin binding and if any of these enzymes are absent, we get dystroglycanopathy. These diseases are challenging to study: one reason being that they clinically present with phenotypic diversity ranging from mild LGMDs to severe MDs with muscle and eye involvement. With this in mind, when studying the disease and its amelioration, we will want an animal model where diverse phenotypes can be rapidly screened, along with potential therapeutics. Barresi and Campbell, Journal of Cell Science, 2006

Zebrafish are advantageous for studying muscle disease Rapid development Conserved genomics Transparent embryos Don’t walk through all of the bullet points. WALK THROUGH MUSCLE FIBERS AND MTJS. Thus, we selected zebrafish as a model organism. In addition to being able to rapidly screen drugs and phenotypes in this model, we can also very easily stain and visualize muscle. Here, a normal, wild-type zebrafish embryo exhibits well-organized, straight muscle fibers stained in red that are anchored to myotendinous junctions in green. Thus, it is very easy to assess and quantify varying degrees of muscle abnormalities, such as dystrophy. So what does dystrophy look like in a zebrafish? Myotendinous junction Muscle fibers

Dystrophic zebrafish modeling dystroglycanopathy exhibit fiber detachment gmppb -/- fktn -/- What does dystrophy look like? Dystrophic zebrafish classically exhibit detached fibers, which will appear as “fiber balls” as outlined by the orange arrows. Here we have two newly identified zebrafish dystroglycanopathy mutants that exhibit various degrees of fiber detachment. Additionally, the FKTN mutant shown here possesses shorter muscle segments. Thus, it is not uncommon to see phenotypic variability in fish models of these diseases. So what about other mutations causing dystroglycanopathy?

Engineering zebrafish mutant models of dystroglycanopathy Founders Identified Founders Growing GMPPB FKTN B3GALnT2 POMGnT2 DPM3 DOLK DPM2 B4GAT1 TMEM5 POMT1 POMT2 POMK POMGnT1 LARGE ISPD At the moment, we have utilized CRISPR/Cas9 mutagenesis to engineer 16 out of the 18 dystroglycanopathies in zebrafish. We have identified 5 founders among our mutant stocks and have additional founders growing. While our mutant stocks have been growing to maturity for identification, we have worked with an FKRP morphant model of secondary dystroglycanopathy. Meanwhile: Use FKRP morpholinos to examine secondary dystroglycanopathy!

FKRP Morphants Exhibit Muscle Defects and Fiber Detachment So in our morphant model, embryos are injected with a gene sensitive oligonucleotide that will prevent translation of FKRP, which plays a critical role in glycosylation. Like you saw a few slides ago, the control zebrafish exhibits well organized muscle with no detachment. FKRP morphants exhibit wider MTJ angles, wavy fibers, and fiber detachment (orange arrows), thus have structural defects. Yet locomotive defects are associated with muscular dystrophies, and we needed to assess that.

Motility is Compromised in FKRP Morphants Therefore, we performed a motility assay in our morphant fish, as well as our controls. Fish were stimulated at the anterior end and we assessed how many touches were required to induce an escape response. Our data demonstrate that it is far more difficult to induce an escape response in morphants, as more touches are required to do so. We would ultimately like to determine the causes of these motility defects. They could be attributed to a structural difference within the muscle. However, it could also be due to disruptions in signals from the nerves. Thus, we also looked at the NMJ. Ct Ct FKRP MO

Neuromuscular Junction Formation is Disrupted in FKRP Morphants How can we ameliorate these abnormalities? Ct FKRP MO Blue arrows: Myoseptal Innervation Orange arrows: Distributed innervation The NMJ as shown here lies at the interface between muscle cells and nerve cells and are essential players in muscle contraction, thus movement. Here, we evaluated zebrafish NMJs in controls and morphants by staining for two NMJ markers: acetylcholine receptors (RED) and synaptic vesicles (GREEN). In controls, both are present and we can see that there is a good balance of myoseptal innervation at the muscle boundaries (BLUE) and distributed innervation (WHITE). In our morphants, we can see that the distributed innervation in particular is significantly reduced, which demonstrates that the NMJ is disrupted in this model and may play a role in reduced motililty. Our data suggest that muscle physiology is highly disrupted in FKRP morphants. So now, how are we going to ameliorate these defects? SV2 α-BTX

NAD+ Rescues Dystrophy and Motility in Dystroglycan Morphants Fiber Detachment Motility Dystroglycan Morphant The first therapeutic we assessed for rescue was NAD+. Our lab previously demonstrated that NAD+ is able to rescue fiber detachment in dystroglycan deficient fish, as we can see a reduction in detachments here. NAD+ also rescued motility. Thus, we asked if NAD+ could also rescue dystrophy in a secondary dystroglycanopathy. Dystroglycan Morphant + NAD+ Goody et al., PLoS Biology, 2012

Exogenous NAD+/Emergen-C Reduces Fiber Detachment in FKRP Morphants FKRP MO + EC Therefore, we treated our morphant fish with Emergen-C, which vitamin precursors to NAD+. We previously demonstrated in the dystroglycan morphants that there is no significant difference between using either approach in terms of rescue. When morphants fish were treated with Emergen-C, we found that muscle fiber organization was substantially improved in most morphants, muscle fibers were thickened, and that fiber detachment was significantly reduced, suggesting a vast improvement in muscle structure. We also assessed how Emergen-C improves muscle function. ** *EC = Emergen-C % myotomes with dystrophy ** + EC

NAD+/Emergen-C Improves Motility and Innervation in FKRP Morphants Ct Blue arrows: Myoseptal Innervation Orange arrows: Distributed innervation SV2 α-BTX EC Which we did via a motility assay and NMJ staining. Our results suggest demonstrate that EC supplementation increases distributed innervation in morphants and we also see an increase in our markers at the muscle segments, which also suggests more myoseptal innervation. Furthermore, the number of touches required to induce an escape response here was significantly reduced in morphants treated with EC. From the data, we can conclude that Emergen-C has therapeutic potential in two dystroglycanopathy models, likely due to the presence of niacin (contains NAD+). FKRP MO FKRP MO + EC *EC = Emergen-C

Addressing phenotypic variability in dystroglycanopathies 2° Dystroglycanopathy Genes POMT1 POMT2 POMGnT1 POMGnT2 LARGE FKTN FKRP ISPD DPM1 DPM2 DPM3 DOLK POMK B3GALNT2 B4GAT1 GMPPB TMEM5 So while we have addressed the therapeutic potential of NAD+ in dystroglycanopathies, we still haven’t assessed the phenotypic variability that occurs with these conditions. There are 18 of these conditions, many of which present with ranges of dystrophy from mild to severe. Our solution to this problem is to utilize our identified dystroglycanopathy mutants. SOLUTION: Utilize identified dystroglycanopathy mutants! Barresi and Campbell, Journal of Cell Science, 2006

Phenotypic Variability in Zebrafish Dystroglycanopathy Mutants SV2 α-BTX We have addressed this variability across a couple of mutant models. As I mentioned earlier, GMPPB and FKTN mutants both exhibit fiber detachment, although the FKTN mutant has more severe muscle defects. Three mutants shown to the left also have varying NMJ defects: B3Galnt2 and pomgnt2 have much reduced distributed innervation, whereas gmppb exhibits distributed innervation that crosses the boundary, which suggests disruptions in myoseptal innervation. We are excited to explore the molecular basis of these varying disruptions across all of our dystroglycanopathy models. Make nmj pictures 2 x 2. Can now say myoseptal is disrupted, distributed goes across, can talk more here about what’s different! White arrow: Myoseptal innervation Arrow heads: Distributed innervation White: Normal Red: Short Green: Long

Zebrafish are a promising preclinical model for dystroglycanopathies Targets: Rescue muscle, motility, AND synaptogenesis Impact: Therapeutics that target the broadest range of muscular dystrophies gmppb -/- fktn -/- In summary, we have demonstrated that NAD+ possesses therapeutic potential in dystroglycanopathy models and that mutant dystroglycanopathy models possess muscle defects and a spectrum of NMJ abnormalities. Ideally, we would like to comprehensively characterize our mutants and test therapeutics that will rescue muscle structure, motility, AND synaptogenesis, with the goal of finding one that will target the broadest range of these diseases. CAH: memorize your transition sentences between each slide, will get point across easier. MFG: get main point across. SV2 α-BTX

Acknowledgements COMMITTEE: HENRY LAB: Clarissa Henry, PhD Rob Wheeler, PhD Greg Cox, PhD Roger Sher, PhD Melissa Maginnis, PhD Mark Nilan (Fish Care) GSBSE, School of Biology and Ecology, March of Dimes, NIH (Financial Support) Wheeler and Kim Labs (Equipment) HENRY LAB: Clarissa Henry, PhD Michelle Goody, PhD Mary Astumian Elisabeth Kilroy Sarah Alrowaished Beth Mason Maggie Pasquarella Joe Belanger And with that, I would like to thank everyone in the Henry lab, particularly our dystroglycanopathy research team, my thesis committee for their insight and support, all departments and agencies that have funded my project, Mark Nilan for fish care, and the Wheeler and Kim labs for equipment usage. Thank you, and I would be happy to take any questions.