Transient Metabolic Stress Induces Energy and Purine Impairment in Neural Progenitor Cells for Lesch-Nyhan Disease Open Access

Grychowski, Lauren (Spring 2024)

Permanent URL: https://etd.library.emory.edu/concern/etds/9w032469m?locale=en
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Abstract

Background: Lesch-Nyhan Disease (LND) is a rare neurological disorder characterized by severe behavioral and motor impairments. LND is caused by pathogenic variants in the HPRT1 gene, which produces an enzyme with a crucial role in purine salvage synthesis. Existing research has yet to examine the impact of acute metabolic stress during neurodevelopment as a potential factor contributing to the pathogenesis of LND. The present study aims to leverage a cell culture model involving neural progenitor cells (NPCs) under conditions that mimic transient glycolytic and purine metabolic stress to determine whether LND cells are more vulnerable to metabolic challenges.

Methods: NPCs were generated from patient-derived induced pluripotent stem cells (iPSCs) and confirmed through immunostaining for neural stem cell markers. Initial experiments were conducted to determine optimal dosage and timing of glycolytic and purine metabolic inhibitors on NPCs to induce metabolic stress while preventing cell death. At these determined conditions, NPCs were again challenged, and purines were quantified utilizing high performance liquid chromatography (HPLC).

Results: Increasing concentrations of glycolytic and purine metabolic inhibitors led to a trend of decreasing ATP levels in both control and LND cell lines. Acute exposure to high concentrations of glycolytic inhibitor resulted in significant differential reduction in total purine content and GTP levels in LND NPCs compared to controls, revealing defects in purine metabolism. Transient exposure to both glycolytic and purine metabolic inhibitors compromised energy metabolism, as demonstrated through reductions in energy transfer purines such as ATP; however, the differences between control and LND NPCs were minimal.

Conclusion: This study suggests that challenging LND NPCs with acute metabolic stressors can reveal impairments in purine and energy metabolism that aren’t evident at baseline, which indicates that this model may be a beneficial tool for investigating LND pathogenesis. Further research is required to identify the optimal conditions and developmental stages of cells that can elucidate the role of energy impairments during LND neurodevelopment. 

Table of Contents

1. Introduction

1. LND Background

4. Purine Metabolism

5. Cell Culture Methods for LND using iPSCs

7. Energetic Impairment Hypothesis

10. Materials and Methods

10. Establishment of iPSC Lines

10. NPC Differentiation and Maintenance

11. Immunostaining

12. Preparation of Inhibitors

12. Bioluminescent ATP Assay

13. Purine Quantification

14. Statistical Analysis

15. Results

15. NPC Validation

15. Reduction of ATP Levels After Inhibitor Exposure in LND NPCs

16. Changes in Purines After 2DG Exposure in LND NPCs

17. Changes in Purines After Azaserine Exposure in LND NPCs

18. Changes in Total Purine Content After Inhibitor Exposure in LND NPCs 

19. Discussion

22. Limitations

23. Future Directions

25. Conclusions

26. Tables and Figures

26. Figure 1: Purine Metabolic Pathway 

27. Table 1: Subject Characteristics 

28. Figure 2: NPC Staining 

29. Figure 3: Cellular ATP Content After 2DG Exposure 

30. Figure 4: Cellular ATP Content After Azaserine Exposure 

31. Figure 5: Concentration of Adenine Derivatives After 2DG Exposure 

32. Figure 6: Concentration of Guanine Derivatives After 2DG Exposure 

33. Figure 7: Concentration of Adenine Derivatives After Azaserine Exposure 

34. Figure 8: Concentration of Guanine Derivatives After Azaserine Exposure 

35. Figure 9: Total Purine Content After Inhibitor Exposure 

36. References

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