GAD: Initiator of Autoimmune Diabetes

1
GAD Initiator of Autoimmune Diabetes
By Reversa Mills
A Review of the The Control of Autoimmune
Diabetes in NOD Mice by GAD Expression or
Suppression in Beta Cells
2
What is Type 1 Diabetes?

• Autoimmune destruction of insulin producing
  pancreatic cells
• Autoantibodies are directed towards specific
  antigenic targets of islet cells
• Destruction of pancreatic beta cells by
  diabetogenic T-cells leads to a deficiency of
  insulin secretion

3
What is GAD?

• Glutamic Acid Decarboxylase(GAD)
• Pancreatic beta cell autoantigen
• Implicated in triggering beta cell-specific
  autoimmune response
• Earliest autoantibody found in pre-diabetic phase
  of human patients
• Presence of anti-GAD antibody is a strong
  predictor of the future development of Type I
  Diabetes

4
Hypothesis of Yoon et al Report

• GAD is the single self protein that initiates
  Autoimmune Diabetes

5
Rationale of Yoon et al Experiments
6
What is NOD mice?

• Non-obese diabetic (NOD) mouse
• A good human type I diabetic model
• GAD provokes earliest T-cell proliferation
  response in NOD mice just as in pre-diabetic
  human patients

7
Experiment 1
8
Design of Antisense GAD Gene

• Construct of antisense GAD transgene for both
  isoforms of rat GAD CDNA (rGAD 65 and rGAD 67)

9
Determination of Successful Transgenic NOD mice
Evaluation at Gene Level

• Reverse PCR was used to quantitate the amount of
  mRNA expressed in the transgenic NOD mice
• cDNA was amplified and separated by gel
  electrophoresis
• Presence of band indicates the animal is an
  antisense GAD transgenic animal

10
Correlation of Successful Transgenic mice from
gel to pedigree

• Purpose of further identification
• Classify as High(H), Medium(M), or Low(L) based
  on intensity of expression of GAD gene
• Designate band or line in the gel with the
  appropriate animal ear tag number

11
Determination of Successful transgenic NOD
miceEvaluation at mRNA level

• Isolated mRNA using a Northern Blot
• 3 type so mRNA evaluated
  (1)AS-GAD 67 (2)AS-GAD 65
  (3)GADPH
• Used a control designated as(-)
• Distinguished 3 types of mice based on the
  intensity of bands (1) High (H)
  darkest (2) Medium (M)
  (3) Low (L) lightest

12
Determination of Successful transgenic NOD
miceEvaluation at protein level

• Characterized GAD expression by using a Western
  Blot
• Complete suppression of beta cell GAD expression
  in islets of H-AS-GAD NOD mice observed compared
  to moderate and low expression in M and L-AS GAD
  NOD mice
• GAD expression identified equally in the brain
  tissues of all 3 lines of AS-GAD NOD mice

13
Determination of Successful Transgenic NOD
miceEvaluation at cellular level

• Immunohistochemical staining of pancreatic islet
  cells
• 3 different types of stains used
  
  (1) HE stain normal stain
  
  (2)Anti-GAD stain- to evaluate GAD expression
  
• (3) Anti-insulin stain- to evaluate insulin
  expression
• All 3 lines of AS-GAD-NOD mice show identical
  expression of insulin to the non-transgenic mice

14
Conclusion of Suppression of Beta cell GAD
Expression
The Results of
Reverse PCR
Immunohistochemical Stain
Northern and Western Blot

• Antisense-GAD mRNA transgenic mice were
  successfully produced
• 3 types of transgenic mice produced
  (1) H-AS-GAD
  (2)
  M-AS-GAD
  (3) L-AS-GAD
• H-AS-GAD NOD mice showed specific Beta cell
  suppression of GAD protein
• All 3 types of transgenic mice had normal insulin
  production

15
Experiment 2
16
Occurrence of Diabetes Relative to GAD Expression

• Graph of diabetes development in 3 lines of
  AS-GAD-NOD mice(Figure C represents the second 3
  lines of AS-GAD-NOD out of the 6 total lines
  selected)
• None of the H-AS-GAD-NOD mice developed diabetes
• Variable percentages of M-and L-AS-GAD NOD mice
  and Non-transgenic mice developed diabetes

17
Morphology of Islet cells in Relation to GAD
Expression

• Comparison of incidence of insulitis due to
  mononuclear infiltration of islet cells (Figure D
  represents the second 3 lines of the AS-GAD-NOD
  out of the 6 total lines selected)
• 80 of the H-AS-GAD NOD islets remained free of
  infiltration
• Moderate to severe insulitis/infiltration was
  observed in M-and L-AS-GAD islet cells and
  significantly in Non-transgenic islet cells

18
Conclusion of Evaluation of Disease Development

• Beta cell GAD expression is required for the
  development of diabetes in NOD mice

19
Experiment 3
20
Does the mere presence of an antisense gene
provide protection against diabetes?

• Yoon designed another antisense transgenic animal
  that carried the antisense endogenous retroviral
  env mRNA
• Endogenous retroviral env protein is also an
  aggressive beta cell autoantigen expressed in
  the beta cells of NOD mice

• Results showed that these antisense transgenic
  mice developed diabetes, at the same rate as the
  non-transgenic mice, despite the fact that the
  antisense transgene was highly expressed and
  blocked the expression of the retroviral env
  protein
• Findings were not consistent with the results
  shown in GAD-suppressed counterparts

21
Conclusion of the SHAM Operation

• Diabetes prevention is not due to a nonspecific
  effect of an antisense transgene

22
Experiment 4
23
Does antisense GAD suppress autoimmunity in other
tissues?
H-AS-GAD TG (-)
Islet Cells
Salivary Glands
24
Conclusion of Evaluation of Antisense GAD
Specificity

• Beta Cell-specific suppression of GAD expression
  by Antisense GAD only affects beta cell-specific
  autoimmunity

25
Experiment 5
26
Effects of GAD protein

• Splenocytes from nondiabetic H-AS-GAD NOD mice
  and nondiabetic non-transgenic mice were
  transfused into severe combined deficiency
  diseased(SCID) NOD mice
• None of the SCID NOD mice that received
  splenocytes from H-AS-GAD NOD mice developed
  diabetes
• 90 of SCID mice that received splenocytes from
  non-transgenic NOD mice developed diabetes

27
Conclusion of GAD protein Mechanism of Action

• GAD protein expression stimulates the generation
  of diabetogenic T-cells

28
Experiment 6
29
Effects of T-cell Proliferation to different
autoantigens

• Examined proliferative response of splenocytes
  from various ages of H-AS-GAD-NOD mice,
  non-transgenic NOD mice, and control NOD mice
• T-cell proliferative responses were measured
  based on amount of radiolabeled thymidine
  precursors
• No significant T-cell proliferation occurred in
  response to any of the autoantigens in the
  H-AS-GAD NOD mice
• Variable amounts of T-cell proliferation occurred
  in response to all of the autoantigens in both
  NOD mice and the non-transgenic NOD mice
• GAD protein generated the greatest T-cell
  proliferative response in both NOD mice and
  non-transgenic NOD mice

30
Conclusion of T-cell Proliferative Response

• Antisense GAD decreases the T-cell immune
  response to other B-cell autoantigens as well as
  blocking T-cell response to GAD

31
Experiment 7
32
Quantitative Analysis of Disease Prevention

• Susceptibility to diabetogenic T-Cell attack was
  measured in acutely diabetic NOD mice following
  their transplantation of either GAD-suppressed
  islets or GAD-expressed islets
• All diabetic NOD mice that received the
  GAD-expressed islets showed a recurrence in the
  development of diabetes
• None of the diabetic NOD mice receiving the
  GAD-suppressed islets developed a recurrence of
  diabetes

33
Morphological Protective Effects of
GAD-Suppressed Beta cells
H-AS-GAD
Islet cells
Islet cells
TG(-)

• Examination of the diabetic NOD mice transplanted
  islet cells
• GAD-expressed transplanted islets (from
  non-transgenic NOD mice) showed massive
  infiltration
• 80 of GAD-suppressed islets (from H-AS-GAD NOD
  mice) remained undamaged and intact

34
Is GAD expression required for Autoimmune
destruction of Beta cells?

• Splenocytes from acutely diabetic NOD mice fused
  into H-AS-GAD NOD and non-transgenic mice
• None of the H-AS-GAD mice developed diabetes
• 71 of non-transgenic mice developed diabetes
• Results show that GAD expression is necessary for
  development of diabetes

35
Conclusion of the Protective Effects of Antisense
GAD

• Beta Cell-specific suppression by antisense GAD
  is effectively capable of preventing the
  development of Autoimmune Diabetes in NOD mice

36
Findings

• The following experiments provided a significant
• correlation between the development of
  diabetes and the
• presence of GAD protein in pancreatic beta
  cells

• Antisense GAD transgenic mice showed effective
• resistance to Diabetes. Therefore, the
  specific absence
• of GAD protein provides immunity to diabetes
  development

• Diabetogenic T-cells develop only in the presence
  of GAD
• protein

• GAD protein is a significant autoantigen that
  initiates
• diabetes by activating GAD-specific
  diabetogenic T-cells