Investigating the role of sphingolipid metabolising enzymes in the pathogenesis of Friedreich’s Ataxia (FRDA)

Abstract

Friedreich’s Ataxia (FRDA, FA) is a neurodegenerative disorder, characterised by a triplet repeat expansion of GAA found within the FXN gene. This leads to reduced transcription and translation of the mitochondrial protein frataxin, which plays a role in mediating iron used as part of the electron transport chain (ETC). This leads to increases in reactive oxygen species (ROS) levels, causing oxidative stress and eventually cell death. This is particularly apparent in the sensory neurons of the dorsal root ganglia (DRG) and the cerebellar dentate nucleus. This also extends to hypertrophic cardiomyopathy and decreased function of the β-cells. The consequences of this for FRDA patients include difficulties in walking, as well as suffering from cardiomyopathy and type 2 diabetes, and patients eventually become reliant on the use of wheelchairs in adulthood. Furthermore, patients often have a shortened lifespan. Symptoms often begin to present themselves around the age of puberty but can be exhibited in children as young as 5 years old. As of October 2024, there is currently no cure for FRDA, so all treatments which are available to patients are palliative. It is therefore crucial that further investigations are carried out to find an effective cure for FRDA. Several treatments have been investigated and some tested in various clinical trials. Current antioxidant therapies have shown to be effective at combatting some of the cardiological symptoms which are present in FRDA, however these have not been successful in targeting the neurological effects of FRDA, and as such, there is a need to find a method of treating FRDA patients which can alleviate these symptoms. In 2023, Omaveloxolone, which acts through the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) became the first FDA approved treatment for FRDA, but it has several limitations, such as no known effect on severe cardiomyopathy, its administration age begins at age 16, it cannot be given to patients with severe hepatic impairment, and it is only approved in the USA and the EU, so it currently cannot be used to treat FRDA patients in the UK. Omaveloxolone remains an incredible starting point for the treatment of FRDA, but further avenues need to be explored to identify if there are possible therapeutic targets which could have a greater effect on the array of symptoms displayed by FRDA patients. Sphingolipids are structural components of cell membranes involved in cell signalling pathways that contribute to cell proliferation and cell apoptosis. Other neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), have been associated with changes to sphingolipids involved in cell apoptosis and cell proliferation. The balance between these different sphingolipids has been well established in cancer, and targeting pro-apoptotic sphingolipids has been investigated in different cancers to determine if this can reduce the progression of cancer. Sphingolipids can be formed through the de novo and salvage pathway synthesis mechanisms, and it has been shown that the sphingolipids ceramide and sphingosine are involved in promoting cell death, and once phosphorylated into ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), respectively, C1P and S1P can then activate pathways which promote cell growth. Previous sphingolipid studies into FRDA have not investigated if there are changes in the levels of the enzymes which regulate sphingolipid synthesis. Therefore, this PhD project has investigated the expression of the enzymes Sphingosine Kinase (SPHK) and Lipid Phosphate Phosphatase (LPP), which directly affect the levels of ceramide, sphingosine, S1P, and C1P, and targeted them, in various FRDA models to identify if there is dysregulation of sphingolipid metabolising enzymes in FRDA and if targeting them improves the mitochondrial dysregulation and reduced frataxin expression which is associated with FRDA. Firstly, the mRNA and protein expression of SPHK and LPP was investigated in healthy control and FRDA human fibroblast cell lines. Experiments showed a significant decrease in SPHK expression and a significant increase in LPP expression in FRDA human fibroblast cell lines when compared to healthy control. Both isoenzymes of SPHK, SPHK1 and SPHK2, and all three isoenzymes of LPP, LPP1, LPP2, and LPP3, were all shown to be changed at the mRNA expression level. These experiments were then repeated in healthy control and FRDA mouse cerebellum tissue, using cerebellum tissue from the Y47R/YG8sR-derived mouse model. Experiments showed a significant decrease in SPHK expression and a significant increase in LPP expression in the YG8sR-derived mouse cerebellum tissue compared to the Y47R. These experiments identified SPHK and LPP as dysregulated enzymes in FRDA models and therefore could be contributing to the sphingolipid changes identified by previous FRDA research. To identify if dysregulation was only of SPHK and LPP or if other sphingolipid metabolising enzymes are also changed in FRDA, the mRNA expression of CDase, CERK, SPP, CerS and S1PR was also measured, with only significant changes identified in CerS1 and CerS2. These results, therefore, identified SPHK and LPP as the most viable enzyme targets within sphingolipid synthesis to take forward for further investigation. The next step after identifying changes in SPHK and LPP expression was to target these two enzymes in healthy control and FRDA human fibroblast cell lines using small molecule compounds and identify if there were any changes to various mitochondrial processes. The two compounds used were K6PC-5, an SPHK agonist, and XY-14, an LPP inhibitor. The optimal concentrations of K6PC-5 and XY-14 were identified and 10 μM K6PC-5, 0.1 μM XY-14 and 1 μM XY-14 were taken forward for investigations into mROS, cellular ROS, DYm, mitochondrial mass, mtDNA copy number, aconitase activity, GSH/GSSG, and H2O2-induced oxidative stress. Treatment with K6PC-5 led to significant improvements in mROS, aconitase activity and GSH/GSSG in FRDA human fibroblast cell lines compared to healthy control. Treatment with XY-14 led to significant improvements in all mitochondrial parameters investigated except for cellular ROS. These results suggested that SPHK and LPP are involved in improving mitochondrial dysfunction. To identify if these compounds exert effects on frataxin and Nrf2, healthy control and FRDA human fibroblast cell lines were treated with K6PC-5 or XY-14 and the frataxin and Nrf2 expression was measured. XY-14 significantly improved Nrf2 and frataxin protein expression in FRDA human fibroblast cell lines compared to healthy control. These results suggest that targeting LPP is more effective at improving mitochondrial dysfunction and frataxin expression than targeting SPHK. Therefore, LPP1 was targeted using shRNA in healthy control and FRDA human fibroblast cell lines, and investigated for the effects on mROS, mtDNA copy number, aconitase activity, Nrf2 expression, and frataxin expression. shRNA-mediated targeting of LPP1 led to significant improvements in all of these experiments apart from Nrf2 expression, which overall indicates the possibility of targeting sphingolipid metabolising enzymes in FRDA could be a viable option. Whilst FRDA human fibroblast cells lines are a useful and popular option amongst FRDA researchers for investigations into new pathways and therapeutics, they are not the most representative model for FRDA research, as the disease is primarily characterised by neuropathy, with cardiomyopathy as a secondary symptom. Therefore, the next step was to investigate SPHK and LPP in more relevant cell types. Human Induced Pluripotent Stem Cells (iPSCs) are a useful way of differentiating stem cells to desired cell types without incurring the ethical issues that embryonic stem cells do. Furthermore, the healthy control and FRDA human fibroblast cell lines used are primary cell lines from various donors, and so there may be other factors which contribute to the results obtained. Therefore, isogenic pairs of healthy control and FRDA human iPSCs were obtained for these experiments. Healthy control and FRDA human iPSCs were differentiated into iPSC-derived cardiomyocytes and iPSC-derived peripheral sensory neurons, and the levels of SPHK and LPP expression were measured. Whilst no changes were found in SPHK expression in the FRDA human iPSC-derived cardiomyocytes, significant changes were found in LPP expression. Significant changes in SPHK and LPP expression were identified in FRDA human iPSC-derived peripheral sensory neurons compared to healthy control. Therefore, the optimal concentrations of K6PC-5 and XY-14 were identified in healthy control and FRDA human iPSC-derived peripheral sensory neurons and 10 μM K6PC-5 and 1 μM XY-14 were taken forward for investigations into mROS, mtDNA copy number, Nrf2 expression, and frataxin expression. XY-14 led to a significant improvement in mtDNA copy number and K6PC-5 treatment significantly improved mROS. However, both K6PC-5 and XY-14 treatment improved frataxin expression in FRDA human iPSC-derived peripheral sensory neurons compared to healthy control. These results suggest that SPHK and LPP dysregulation is found in FRDA human iPSCderived cardiomyocytes and FRDA human iPSC-derived peripheral sensory neurons, and that targeting them may have some improvements. Overall, the results in this thesis have shown SPHK and LPP dysregulation in a number of different FRDA models and have shown that targeting SPHK and LPP may have some effect on both frataxin expression and FRDA-associated mitochondrial dysfunction. Future investigations in FRDA animal models could identify if targeting SPHK and LPP could have any therapeutic effect on the difficulties with balance and coordination which FRDA patients face.