1. Determining the Neuron Type Specificity of a Potential Mechanism for Alcohol Addiction
Dalton Huey

2. Alcohol Use Disorders (AUD)
Compulsive craving and consumption of alcohol
NAAA stats

3. Alcohol Use Disorders (AUD)
Compulsive craving and consumption of alcohol
4th leading cause of preventable death in the United States

4. Alcohol Use Disorders (AUD)
Compulsive craving and consumption of alcohol
4th leading cause of preventable death in the United States
Heritability: 40-60% of risk for developing AUD

5. Ethanol Responsive Genes
Gene expression in response to ethanol
Wolen et al. (2012)

6. Ethanol Responsive Genes
Gene expression in response to ethanol High response to ethanol
Wolen et al. (2012)

7. Ethanol Responsive Genes
Gene expression in response to ethanol High response to ethanol High correlation with other genes
Wolen et al. (2012)

8. Ethanol Responsive Genes
Gene expression in response to ethanol High response to ethanol High correlation with other genes
Gsk3b
Wolen et al. (2012)

9. Glycogen Synthase Kinase 3 Beta (GSK3B)
Serine/Threonine Kinase

10. Glycogen Synthase Kinase 3 Beta (GSK3B)
Serine/Threonine Kinase
Multifunctional

11. Glycogen Synthase Kinase 3 Beta (GSK3B)
Serine/Threonine Kinase
Multifunctional
Involved in ethanol induced neurotoxicity

12. Does Gsk3b Influence Alcohol Consumption?
Experiment:
Gsk3b overexpressed in neurons
Alcohol consumption measured

13. Does Gsk3b Influence Alcohol Consumption?
Experiment:
Gsk3b overexpressed in neurons
Alcohol consumption measured
van der Vaart et al. (2018)

14. Does Gsk3b Influence Alcohol Consumption?
Experiment:
Gsk3b overexpressed in neurons
Alcohol consumption measured
van der Vaart et al. (2018)

15. Which Genes Are Influenced by Gsk3b?
Experiment:
Gsk3b overexpressed in neurons
Transcript levels measured for synaptic signaling genes

16. Which Genes Are Influenced by Gsk3b?
Experiment:
Gsk3b overexpressed in neurons
Transcript levels measured for synaptic signaling genes
van der Vaart et al. (2018)

17. Brain Derived Neurotrophic Factor (BDNF)

18. Brain Derived Neurotrophic Factor (BDNF)

19. How does increasing GSK3B increase ethanol consumption?
Possible Mechanism
How does increasing GSK3B increase ethanol consumption?

20. How does increasing GSK3B increase ethanol consumption?
Possible Mechanism
How does increasing GSK3B increase ethanol consumption?

21. How does increasing GSK3B increase ethanol consumption?
Possible Mechanism
How does increasing GSK3B increase ethanol consumption?
Which Neurons?

22. Camk2a+ Neurons
Camk2a – Calmodulin Dependent Protein Kinase 2 Alpha

23. Camk2a+ Neurons Camk2a – Calmodulin Dependent Protein Kinase 2 Alpha
Only in glutamatergic pyramidal neurons in the forebrain

24. Camk2a+ Neurons Camk2a – Calmodulin Dependent Protein Kinase 2 Alpha
Only in glutamatergic pyramidal neurons in the forebrain

25. Camk2a+ Neurons Camk2a – Calmodulin Dependent Protein Kinase 2 Alpha
Only in glutamatergic pyramidal neurons in the forebrain

26. Camk2a+ Neurons Camk2a – Calmodulin Dependent Protein Kinase 2 Alpha
Only in glutamatergic pyramidal neurons in the forebrain
Likely Candidate Neuron Type to Explain Gsk3b Effects

27. Main Question:
All Neurons

28. Main Question: Is this effect dependent on only the Camk2a+ neurons?
All Neurons
Is this effect dependent on only the Camk2a+ neurons?

29. Experiment Overview
Process:

30. Experiment Overview Process:
1. Overexpress Gsk3b only in Camk2a+ neurons

31. Experiment Overview Process:
1. Overexpress Gsk3b only in Camk2a+ neurons
2. Measure expression levels of Bdnf

32. Experiment Overview Process:
1. Overexpress Gsk3b only in Camk2a+ neurons
2. Measure expression levels of Bdnf
3. Compare against controls

33. Cre-Dependent Overexpression of Gsk3b
Gsk3b gene in reverse orientation

34. Cre-Dependent Overexpression of Gsk3b
Gsk3b gene in reverse orientation

35. Cre-Dependent Overexpression of Gsk3b
Gsk3b gene in reverse orientation
Cre-recombinase enzyme flips Gsk3b

36. Cre-Dependent Overexpression of Gsk3b
Gsk3b gene in reverse orientation
Cre-recombinase enzyme flips Gsk3b
GSK3B produced only where Cre is active
Mention that it will be a plasmid

37. Overexpression in Camk2a+ Neurons

38. Overexpression in Camk2a+ Neurons
Virus

39. Overexpression in Camk2a+ Neurons
Virus

40. Overexpression in Camk2a+ Neurons
Virus

41. Overexpression in Camk2a+ Neurons
Virus

42. Overexpression in Camk2a+ Neurons
Virus

43. Overexpression in Camk2a+ Neurons
Virus

44. Overexpression in Camk2a+ Neurons
Virus

45. Overexpression in Camk2a+ Neurons
Virus

46. Overexpression in Camk2a+ Neurons
Virus

47. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

48. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

49. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

50. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

51. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

52. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

53. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

54. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

55. Measuring Bdnf Expression Levels
Real Time Quantitative Polymerase Chain Reaction (RT-qPCR) Ct Ct

56. Comparison against Controls
Overexpression in Camk2a+ Neurons
Virus
Virus
Virus

57. Comparison against Controls
Overexpression in Camk2a+ Neurons
Virus
Overexpression in all Neurons
Virus
Virus

58. Comparison against Controls
Overexpression in Camk2a+ Neurons
Virus
Overexpression in all Neurons
Virus
No Overexpression
Virus

59. Possible Results

60. Possible Results
Controls
No Gene
Neuronal
Camk2a+

61. Possible Results
Bdnf Downregulation Fully Dependent on Camk2a+ Neurons
No Gene
Neuronal
Camk2a+

62. Possible Results
Bdnf Downregulation Partially Dependent on Camk2a+ Neurons
No Gene
Neuronal
Camk2a+

63. Possible Results Bdnf Downregulation Not Dependent on Camk2a+ Neurons
No Gene
Neuronal
Camk2a+

64. Implications
Better understanding of ethanol pathway

65. Implications Better understanding of ethanol pathway
Better evaluation of Gsk3b as a therapeutic target for AUD

66. Questions?

68. Supplementary Information

69. Gsk3b Deletion in Camk2a+ Neurons
Decreased Ethanol Consumption

70. Synthesizing the Plasmid – Gibson Assembly

71. Synthesizing the Plasmid – Gibson Assembly

72. Synthesizing the Plasmid – Gibson Assembly

73. Synthesizing the Plasmid – Gibson Assembly