Transient Metabolic Stress Induces Energy and Purine Impairment in Neural Progenitor Cells for Lesch-Nyhan Disease Open Access
Grychowski, Lauren (Spring 2024)
Published
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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