(October 27, 2021) Webinar: GSK3 Inhibition and Muscle Wasting Disorders: Muscular Dystrophy and Spaceflight

1. GSK3 Inhibition and Muscle Wasting Disorders: Muscular Dystrophy and Spaceflight Val Fajardo Ph.D. Canada Research Chair in Tissue Remodeling and Remodeling and Plasticity Brock University

2. Twitter: @MPHDLab 2 Ryan Baranowski MSc student KennedyWhitley MSc student Sophie Hamstra PhD student Sebastian Silvera Former Lab Technician Muscular dystrophy Spaceflight: NASA Jessica Braun PhD student

3. Today’s talk 1. GSK3 – why are we interested in it? 2. GSK3 inhibition and Duchenne muscular dystrophy  **Cognitive decline – highlight work from Dr. Rebecca MacPherson 3. GSK3 and spaceflight: a potential therapeutic target? Twitter: @MPHDLab 3

4. Glycogen synthase kinase 3  Glycogen synthase regulator  Ser/Thr kinase that has over 100 substrates  Two isoforms (GSK3⍺ and GSK3β)  Dominant isoform in muscle Rippin et al., (2021) Cells 10(2): 262. Twitter: @MPHDLab 4

5. Why are we interested in GSK3  Inhibiting this enzyme in muscle can: 1. ↑ muscle size/mass Twitter: @MPHDLab 5

6. Twitter: @MPHDLab 6 Rommel et al., 2001 Nat. Cell Biology Vyas et al., 2002AJPCell Physiology

7. Nigel Kurgan PhD Candidate Supervisor: Dr. Nota Klentrou Kurgan et al., (2019) Cells 8(11):1340 Twitter: @MPHDLab 7

8. Why are we interested in GSK3  Inhibiting this enzyme in muscle can: 1. ↑ muscle size/mass (various mechanisms: ↑ protein synthesis, ↓ protein breakdown, ↑ myoblast fusion and myonuclear accretion) 2. ↑ muscle strength Twitter: @MPHDLab 8

9. Twitter: @MPHDLab 9 KennedyWhitley MSc student GSK3 and mdx muscle pathology Whitley et al., (2020) Physiological Reports 8:e14517 Soleus EDL • Fed 3-6 month old male mice lithium (10 mg/kg/day) for 6 weeks • Low dose 0.02 mM circulating Li • Common dose with bipolar disorder = 0.5 mM Li

10. Why are we interested in GSK3  Inhibiting this enzyme in muscle can: 1. ↑ muscle size/mass (various mechanisms: ↑ protein synthesis, ↓ protein breakdown, ↑ myoblast fusion and myonuclear accretion) 2. ↑ muscle strength 3. ↑ promote the oxidative fibre type and fatigue resistance Twitter: @MPHDLab 10

11. Twitter: @MPHDLab 11  Muscles are heterogenous and are comprised of several different fibre types IIA I IIX IIB Fibre type vehicle tideglusib

12. Twitter: @MPHDLab 12 IIA I IIX IIB Fibre type Contractile kinetics (speed of contraction and relaxation) slowest fastest Metabolism (ATP production) oxidative glycolytic Fatigability least most Contractile and metabolic properties Calcineurin signaling promotes the oxidative fibre type by activating NFAT

13. Twitter: @MPHDLab 13 Vyas et al., 2002AJPCell Physiology ↑ Oxidative myogenic program • GSK3 represses calcineurin/NFAT signaling • Inhibition of GSK3 should promote the oxidative fibre type

14. Lithium and murine soleus remodelling Kennedy Whitley MSc student GSK3 and mdx muscle pathology Whitley et al., (2020) Physiological Reports 8:e14517 Twitter: @MPHDLab 14

15. Why are we interested in GSK3  Inhibiting this enzyme in muscle can: 1. ↑ muscle size/mass 2. ↑ muscle strength 3. ↑ promote the oxidative fibre type and fatigue resistance 4. Improve calcium regulation (SERCA pump) Twitter: @MPHDLab 15

16. Twitter: @MPHDLab 16 GSK3 inhibition can improve SERCA function Thank you Aurora Scientific for this video!

17. Hamstra et al., 2020 Exp. Physiology Twitter: @MPHDLab 17 Control LiCl Control LiCl SERCA2a - PLN - Sophie Hamstra PhD student Increasing calcium concentration

18. Hamstra et al., 2020 AJP Cell Physiology Twitter: @MPHDLab 18 Sophie Hamstra PhD student mdx mouse – DMD model

19. Recap: why are we interested in GSK3  Inhibiting this enzyme in muscle can: 1. ↑ muscle size/mass 2. ↑ muscle strength 3. ↑ promote the oxidative fibre type and fatigue resistance 4. Improve calcium regulation (SERCA pump) Bottom line: All of the above are important factors in conditions of conditions of muscle wasting! Twitter: @MPHDLab 19

20. Today’s talk GSK3 – why are we interested in it? 2. GSK3 inhibition and Duchenne muscular dystrophy  **Cognitive decline – highlight work from Dr. Rebecca MacPherson 3. GSK3 and spaceflight: a potential therapeutic target? Twitter: @MPHDLab 20

21. Twitter: @MPHDLab 21

22. Research objective: slow DMD progression via GSK3 inhibition 1. ↑ Muscle mass 2. ↑ Muscle strength 3. ↑ Oxidative phenotype 4. Enhance calcium regulation Twitter: @MPHDLab 22

23. D2 mdx mouse: a more severe model Twitter: @MPHDLab 23 van Putten et al., 2019 FASEB J 10 weeks old

24. D2 mdx mouse: a more severe model Twitter: @MPHDLab 24 Coley et al., 2016 Hum Mol Genetics

25. Twitter: @MPHDLab 25 KennedyWhitley MSc student GSK3 and mdx muscle pathology Whitley et al., unpublished Keypoint: The D2 strain have more GSK3 and the GSK3 that is there is more active!

26. Inhibit GSK3 with tideglusib  Potent non-ATP competitive GSK3 inhibitor  Most clinically advanced  Alzheimer’s  Diabetes  Myotonic dystrophy  Rapid clinical integration?? Twitter: @MPHDLab 26

27. D2 mdx mouse: tideglusib treatment Twitter: @MPHDLab 27 KennedyWhitley MSc student GSK3 and mdx muscle pathology Whitley et al., unpublished  10 mg/kg/day (oral gavage)  2-4 weeks  Age: 6-7 weeks at start of treatment Sebastian Silvera Previous Lab Technician

28. 1.Tideglusib & mdx muscle mass Twitter: @MPHDLab 28 W T m dx-vehicle m dx-tideglusib 0.9 1.0 1.1 1.2 1.3 1.4 1.5 Fold-change in lean mass 0.0005 0.0446 Whitley et al., unpublished iNSiGHT DXA

29. 2.Tideglusib and mdx muscle strength Twitter: @MPHDLab 29 0 500 1000 1500 Impulse (s*g) A WT mdx-vehicle mdx-tideglusib 0 50 100 150 0 50 100 150 Frequency (Hz) Force (mN/mm 2 ) mdx-vehicle mdx-tideglusib WT ** * **** **** **** **** #### #### #### #### * B 0 20 40 60 50% rundown time (s) C Whitley et al., unpublished

30. 3.Tideglusib and fibre type composition Twitter: @MPHDLab 30 Whitley et al., unpublished

32. Tideglusib and mdx histopathology and utrophin Twitter: @MPHDLab 32 Whitley et al., unpublished

33. Tideglusib and serum CK (marker of muscle damage) Twitter: @MPHDLab 33 Whitley et al., unpublished

34. 4.Tideglusib and cardiac calcium regulation Twitter: @MPHDLab 34 Sophie Hamstra PhD student Hamstra et al., unpublished

35. 4.Tideglusib and cardiac calcium regulation Twitter: @MPHDLab 35 Sophie Hamstra PhD student Hamstra et al., unpublished

36. Tideglusib and HF-ultrasound Twitter: @MPHDLab 36 Hamstra et al., unpublished Scintica ProspectT1 Tonya Coulthard Consistent with Coley et al., 2016 Impairments in diastolic function are blunted with tideglusib

37. Recap: GSK3 inhibition in mdx mice  ↑ muscle size and gains in lean mass  ↑ muscle strength and fatigue resistance  ↑ the oxidative fibre type and attenuated muscle damage  Improved SERCA function and mitigated the diastolic dysfunction  Future work: long-term tideglusib treatment  Is it well-tolerated?  Is it more effective?  Can it prevent cardiac dysfunction and prolong lifespan?  Prevent cognitive decline?** Twitter: @MPHDLab 37

38. Alzheimer’s like pathology in the mdx brain Twitter: @MPHDLab 38 • Cognitive impairments are often reported in patients with DMD • Mechanisms remain unknown

39. GSK3 and Alzheimer’s disease Twitter: @MPHDLab 39

40. Today’s talk GSK3 – why are we interested in it? GSK3 inhibition and Duchenne muscular dystrophy  **Cognitive decline – highlight work from Dr. Rebecca MacPherson 3. GSK3 and spaceflight: a potential therapeutic target? Twitter: @MPHDLab 40

42. What can GSK3 inhibition offer?  Muscle fibre type  Muscle atrophy?  Muscle strength?  Calcium regulation? Twitter: @MPHDLab 42

43. Hindlimb suspension  Hindlimb suspension: best accepted spaceflight analog Twitter: @MPHDLab 43 1. Hindlimb unloading ↓ MHC I 2. Hindlimb unloading ↑ GSK3 activation Inhibitory serine GSK3 phosphorylation  7 days of suspension  Rat soleus  ATP competitive GSK3 inhibitorAR-A014418 Sharlo et al., 2018 Biochemistry: Membrane & Cell Bio 12(1): 85-91; Drenning et al., (2008) AJP – Cell Physiol 294:C1088-C1095

44. What about atrophy and weakness? Objectives  2 weeks of unloading  Muscle and bone mass  Targeted DXA (iNSiGHT)  Fibre type composition and size analysis  Muscle strength and fatigue resistance Twitter: @MPHDLab 44 Ryan Baranowski MSc student  Hindlimb suspension: GSK3 (alpha and beta) knockdown

45. What about calcium regulation?  SERCA pumps are impaired in soleus muscles from male and female spaceflown mice (can GSK3 inhibition mitigate this?) Twitter: @MPHDLab 45 Jessica Braun PhD student Male soleus 35 days Female soleus 37 days ATP ATP Braun et al., Int. J Mol. Sciences (In press)

46. Thank you!! Twitter: @MPHDLab 46

47. Q&A Session WWW.SCINTICA.COM INFO@SCINTICA.COM Please enter your questions in the Q&A section. Thank You!

(October 27, 2021) Webinar: GSK3 Inhibition and Muscle Wasting Disorders: Muscular Dystrophy and Spaceflight

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