1. 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

2. 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…

3. 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

4. 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

5. 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

6. 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?

7. 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!

8. 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.

9. 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

10. 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

11. 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

12. 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

13. NAD+/Emergen-C Improves Motility and Innervation in FKRP MorphantsCt
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

14. 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

15. 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

16. 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

17. 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.