EFFECTS OF A PADI2 INHIBITOR DURING ZEBRAFISH MYELINATION

Sarah Shapley Neurobiology Senior Baldwin Wallace University April 1st, 2020 CNS MYELINATION CAN BE VISUALIZED IN VIVO

Zebrafish myelination over 10-hour time span

Green: protein

Red: polymerized, F-actin

Nawaz et al., 2015 2 MBP STABILIZES MYELIN SHEATH LAYERS DURING COMPACTION

MBP

Leading edge

Axon

3 HOW IS PADI2 INVOLVED IN WRAPPING DURING MYELINATION?

Research Question

4 PADi2 is increased in MS • Citrullination of myelin proteins increases 4-fold during • Inhibiting peptidyl deiminase (PAD) activity may stabilize myelin sheath in MS (Wood et al., 2013)

5 PADI2 IS EXPRESSED MYELINATING OLIGODENDROCYTES

PADi2 expression

• PADi2 may have increased activity in MS due to remyelination of inactive lesions

Fragments per kilobase/million reads* *Standardized method to quantify expression reading RNA-seq data 6 PADI2 CITRULLINATES ACTIN, TO PREVENT POLYMERIZATION DURING MYELINATION

Hypothesis

7 Expected Results • If actin is citrullinated during wrapping, then g-actin > f-actin • g/f = large/small = LARGE number • Ex: g = 10,000, f = 1,000 then g/f = 10

More g-actin

Fig 7. Zuchero et al., 2015

8 Expected Results

• If citrullination is inhibited during wrapping, then g-actin < f-actin • g/f = small/large = SMALL number • Ex: g = 1,000, f = 10,000 then g/f = 0.1

More f-actin

XX

Fig 7. Zuchero et al., 2015

9 WILD-TYPE ZEBRAFISH

1. Breed WT fish for embryos 2. Grow fish up to designated time point 3. 12 h drug or vehicle exposure --0.2 % DMSO (Vehicle control) -- 1 µM pan-PAD inhibitor 4. Homogenize fish and collect all proteins for G/F-actin assay

10 60 hpf larvae G/F-actin ratios are not significantly affected by the pan-PAD inhibitor

11 PADI2 CITRULLINATES MBP, TO PREVENT PRE-MATURE COMPACTION

Hypothesis

12 VARIATIONS OF MYELIN SHEATH THICKNESS

• More wrapping around the axon • Conduction speed is faster Thick myelin

• Less wrapping Thin myelin • Conduction speed is slower • Leaking ions 13 TRANSGENIC ZEBRAFISH

1. Breed transgenic fish for embryos construct = Tg(mbp:EGFP-CAAX) (Almeida, 2011). 2. Grow fish up to designated time point 3. 12 h drug or vehicle exposure -0.2% DMSO (Vehicle control) -1 µM pan-PAD inhibitor 4. Image using a confocal microscope mbp gfp 14 PADi2 is involved in oligodendrocyte differentiation • Inhibiting myelinating oligodendrocytes • Possible differentiation effects at 48-60 hpf times • Count oligodendrocytes within myotomes of zebrafish post-drug exposure

8 p = 0.23 7 6 5 4 per fish Average 3 2 1

(Oligodendrocyte/myotome) (Oligodendrocyte/myotome) 0 2CA DMSO LIVE IMAGING

• Image myelin in the dorsal and ventral nerve cord

16 At 5 dpf, 2-CA treated zebrafish did not have differences in myelination compared to DMSO 2-CA Tx, n = 6 40 p = 0.187 35 30 intensity 25 DMSO Tx, n = 4 20 15

fluorescence fluorescence 10 5 Mean 0 2CA DMSO

17 IN CONCLUSION… • Future directions/questions • Electron microscopy for myelin thickness • Conduct actin assay in CNS tissue rather than whole homogenates • Combat this through immunoprecipitation known citrullinated myelin proteins (MBP, MOG, MAG) • PADi2 inhibitors may not make effective MS treatments due to inhibiting active remyelination

18 ACKNOWLEDGEMENTS • BWU Biology Dept and Neuroscience Dept • Dr. Morris • Dr. Barko • Gregg DiNuoscio • Dr. Kucenas and Dr. Fontenas (University of Virginia), NeoMed, Oberlin • Friends and Family Funding • Nu Rho Psi Undergraduate Research Grant (2019) • BWU Lucille & Kenneth McCauliffe Grant & Scholarship (2018) • Ohio Space Grant Consortium Scholarship (2018, 2019) • National Institutes of Health Undergraduate Scholarship Program, UGSP (2018, 2019) 19 QUESTIONS?

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