Chemie | Biochemie | Medizin
Nico Hänggi, 2003 | Olten, SO
Parkinson’s Disease is a neurodegenerative disease affecting dopaminergic neurons (DA neurons) and its phenotype includes neuroinflammation. GBA1 mutations increase the risk of Parkinson’s Disease significantly. The endolysosomal storage disorder caused by GBA1 mutations has been shown to trigger Ca2+ release from the ER and lysosomes via Ryanodine Receptors (RyR). These experiments investigated the effects of RyR stimulation on DA neurons generated from human induced Pluripotent Stem Cells (iPSCs). The expression of RyR forms (RYR1, RYR2, RYR3) was analysed by non-quantitative PCR. The effects of the agonists on cell cultures containing iPSC-derived mesencephalic neurons were studied at different concentrations of RyR-agonists and analysed with a qPCR assay for TNFa and COX-2 and with immunostaining for pan-neuronal markers (MAP2/bTUBIII) and a DA neuron marker (TH). This pilot study suggests the agonists led to an upregulation of TNFa and a change in COX-2 expression. The immunostaining showed that DA neurons were heavily affected by treatment because soma count and the number of processes were reduced. Thus, RyR agonist treatment seems to negatively affects the health of neurons and DA neurons and to upregulate the expression of neuroinflammatory genes.
This thesis aimed at answering the following questions: (i) which forms of Ryanodine Receptors are expressed in cell cultures containing iPSC-derived mesencephalic neurons? (ii) how do the cell cultures react to treating them with Ryanodine Receptor agonists?
To answer (i), the expression of the genes RYR1, RYR2 and RYR3 in the cell cultures was analysed with non-quantitative PCR assays using RNA isolates from the cell cultures. For (ii), the cell cultures were treated with the Ryanodine Receptor agonists Suramin and 4-Chloro-m-cresol for 72 hours. After the treatment, RNA levels of TNFa and COX-2 were analysed with qPCR assays for the genes TNF and PTGS2 using RNA isolates from the cell cultures before and after treatment. The effects of treatment on cell count and cell health were analysed with immunostainings for pan-neuronal markers (MAP2, bTUBIII) and DA neuron markers (TH).
The analysis for (i) showed that the genes RYR1, RYR2 and RYR3 are expressed in the cell cultures. The analyses for (ii) showed that treated cell cultures exhibit increased RNA levels of TNFa by 1.86x to 5.13x and the levels of COX-2 were altered -2.2x to 1.3x. The immunostainings showed a non-quantified but apparent reduction in the cell count, shorter and fewer processes and an overall less healthy appearance of the treated DA neurons. The treatment of neurons with 4-Chloro-m-cresol showed the same effect as the treatment of DA neurons with Suramin or 4-Chloro-m-cresol. Suramin treatment did not affect the neurons. Overall, the effect of the agonist treatment was more pronounced on DA neurons than on neurons.
Interestingly, the analysis for (i) showed that all 3 forms of Ryanodine Receptors are expressed in the cell cultures. This is contrary to the literature and might be explained by the heterogeneity of the cell cultures of which only a share are neurons and DA neurons. For (ii), the experiments showed that treating DA neurons with Ryandodine Receptor agonists negatively affects the health of DA neurons and might cause cell death. The treatment also negatively affects neurons but the effect on DA neurons is stronger. The results of the qPCR also suggest a pro-inflammatory effect of Ryanodine Receptor agonist treatment even though for these experiments the caveat of the heterogeneity of the cell cultures applies.
This pilot study managed to show likely negative and pro-inflammatory effects of Ryanodine agonist treatment on DA neurons. To confirm these observed effects, more experiments and analyses would be needed. If the observed tendency was consistent with further experiments, mutant GBA1-induced Glucosylceramide malfunction would be a putative factor for neuroinflammation of DA neurons and would thereby be a putative factor for Parkinson’s Disease. This mechanism could be a target for future treatment of Parkinson’s Disease.
Würdigung durch den Experten
Prof. Beat Suter
Nico Hänggi zeigt ein sehr beachtliches Verständnis für sein Pilotprojekt, welches mit Hilfe von induzierten pluripotenten Stammzellen untersucht, wie Fehlregulation von neuronalen Aktivitäten zu Neurodegeneration, wie z.B. Parkinson’s Disease, führen kann. Er zeigt eine reife, kritische Auseinandersetzung mit den Experimenten und deren Resultaten. Die ersten Immunfärbungen deuten auf einen starken Effekt der getesteten Ryanodine Receptor Agonisten auf die dopaminergen Neuronen hin. Nach der Behandlung waren weniger Neuronen dieser Art vorhanden, und diese produzierten auch weniger Neuriten.
Sonderpreis «MILSET International Science Summer Camp (ISSC)» gestiftet von der SJf-Trägerschaft
Lehrer: Dr. Alexander Rauch