Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells

1. Control of autoimmune diabetes in NOD mice by GAD expression or suppression in beta cells Presented by Kendra Sipres Ji-Won Yoon, Chang-Soon Yoon, Hye-Won Lim, Qi Quan Huang, Yup Kang, Kwang Ho Pyun, Kensuke Hirasawa, Robert S. Sherwin, Hee-Sook Jun

2. INTRODUCTION • Diabetes effects 1 out of 400-500 people • Three types of diabetes • Type 1 diabetes • Type 2 diabetes • Gestational diabetes

3. TYPE 1 DIABETES • Islets of Langerhans produce insulin • Control glucose levels in blood stream • Islets destroyed in type 1 diabetes • Inability to transport sugar in body • Not enough energy in body • pH levels become acidic

4. RATIONALE • Is the expression of GAD required for the development of diabetes in NOD mice? • The effect of GAD suppression in NOD mice • The effect of GAD suppression on autoimmunity

5. GAD • Glutamic acid decarboxylase • Expressed in people and NOD mice • Suppression = prevention • Presence = diabetes development • 2 forms of GAD • GAD 65 • GAD 67

6. NOD MICE • Transgenic mouse • Created to suppress GAD expression • Neither GAD isoform expressed • Considered the best animal model for human diabetes • Rat Gad under control of rat insulin promoter (RIP) • rGAD65 • rGAd67

7. TRANSGENIC NOD MICE • 6 lines of mice established • 1st three lines • H-AS-GAD-NOD • M-AS-GAD-NOD • L-AS-GAD-NOD • 2nd three lines • HK-AS-GAD-NOD • MK-AS-GAD-NOD • LK-AS-GAD-NOD

8. DATA RIP ANTISENSE STRUCTURE DETERMINATION AND AMOUNT OF MICE WITH TRANSGENE EXPRESSION NORTHERN BLOT ANALYSIS TEST PROTEIN IMMUNOBLOT ANALYSIS ISLET IMMUNOHISTOCHEMICAL STAINING

9. INTERPRETING the DATA • Figure B & C • intensity of bands determine which lines were H,M,L • Figure D – northern blot analysis • H band was strongest, followed by M, then L • H band had strongest GAD suppression • Figure E – protein immunoblot analysis • GAD expressed in negative littermates and L • GAD expressed in all groups in the brain • Figure F – immunohistochemical staining • Islets stained

10. DISEASE DEVELOPMENT • Disease development observed in 3 lines of AS-GAD-NOD mice & (-) littermates at 40 weeks old • H-AS-GAD-NOD • 0% developed diabetes • M-AS-GAD-NOD • 67% developed diabetes • L-AS-GAD-NOD • 75% developed diabetes • Transgene negative littermantes • 81% developed diabetes

11. ISLET TISSUE at 20 weeks old • H-AS-GAD-NOD • 80% intact, 20% show periinsulitis • M-AS-GAD-NOD • <20% intact, medium to severe damage, insulitis • L-AS-GAD-NOD • <10% intact, severe damage, insulitis • TRANSGENE NEGATIVE LITTERMATES • <10% intact, severe damage, insulitis

12. DISEASE DEVELOPMENT(2nd 3 LINES OF AS-GAD-NOD MICE) • Hk-AS-GAD-NOD • 2.8% developed diabetes • Mk-AS-GAD-NOD • 83.3% developed diabetes • Lk-AS-GAD-NOD • 80.8% developed diabetes • TRANSGENE NEGATIVE LITTERMATES • 85.7% developed diabetes

13. RATIONALE (2) • Do beta cell-specific suppression of Gad expression affect beta cell-specific autoimmunity? • Does the suppression of GAD expression in beta cells inhibit disease development? • Does it block the generation of Beta cell-specific diabetogenic T cells?

14. Experiment Results • Salivary glands tested • Results indicate autoimmunity isn’t affected • Splenocytes transfused in NOD.scid mice • Results indicate ability to transfer diabetes is blocked in absence of GAD expression

15. CONCLUSION • The data that was collected demonstrated that the GAD expression is required for autoimmune destruction of beta cells • Further research is needed • Hopefully a cure is in the near future