A Biochemical Investigation into the Neuropathology of Cystinosis
Aim: Cystinosis is a rare genetic disorder characterised by excessive intralysosomal cystine accumulation. This lysosomal storage disease is known to cause abnormal brain development and late-onset neurological deterioration, yet the mechanism is poorly understood. Therefore, the aim of this project was to investigate the molecular mechanisms underpinning neuropathology in cystinosis, using C6 glioma and bone-marrow derived macrophage (BMDM) cell lines as a model of brain glial cells. Results: Cystine dimethylester (CDME) and lipopolysaccharide (LPS) were used to mimic the conditions of cystinosis and inflammation in vitro, respectively. Pre-incubation of cells with 500 µM CDME had no effect on the rate of L-[3H]glutamate transport in the absence of sodium ions, whereas 100 ng/µL LPS significantly increased uptake (321% of control) in BMDMs. In contrast, sodium-dependent transport of 200 µM L-[3H]glutamate was significantly reduced following pre-incubation with 500 µM CDME (80% of control and 74% of LPS-treated cells) in C6 glioma cells and 58% of LPS-treated BMDMs. Pre-incubation of BMDMs with 500 µM CDME and 100 ng/µL LPS also caused a significant increase in intracellular glutathione (GSH) (129.9% and 146.7% of control, respectively), but a significant decrease in intracellular GSH (84.6% of LPS-treated cells) was observed following CDME loading. Additionally, CTNS gene silencing was successfully used to generate an alternative in vitro model of cystinosis in C6 glioma cells. Conclusion: For the first time, this study has shown that CDME causes a disruption in the activity of the high-affinity glutamate transporters in vitro, an effect that increases following LPS-activation, whilst CTNS gene silencing provides an alternative in vitro model of the disease. Therefore, this study forms the basis for future investigations into the molecular pathogenesis of progressive neurological deterioration in cystinosis.