Clinical Trials

Objective: To generate and characterize induced pluripotent stem cells (iPSCs)-derived in vitro cellular models of Autosomal Recessive Juvenile Parkinsonism (ARJP) carrying Park2 mutations with a focus on the study of glutamate toxicity. Background: Park2 encodes for a ubiquitin-ligase protein whose mutation upregulates Gluk2, a subunit of the glutamate kainate receptor (KAR) [1], expressed in astrocytes and SNpc neurons. KAR upregulation may induce neuronal and glial excitotoxicity, leading to an early synaptopathy, neuroinflamation and consequently neurodegeneration. Method: Fibroblasts and lymphocytes from Park2 patients and age-matched controls were reprogrammed into iPSCs and further differentiated into dopaminergic neurons [2] and astrocytes [3]. Mesencephalic organoids were also generated and differentiated [4]. Protein expression profile was analysed through western blot (WB), qPCR and immunofluorescence (IF). Electrophysiology assessment was performed on dopaminergic neurons and midbrain organoids in order to better functionally profile these models. Results: We found Gluk2 significantly overexpressed in PARK2 midbrain organoids and astrocytes. Calcium-imaging and HD-MEAs showed an oscillatory augmented reactivity in PARK2 midbrain organoids. We also found a significant tyrosine hydroxylase (TH) overexpression in PARK2 dopaminergic neurons and midbrain organoids and a reactive astrocytosis in PARK2 astrocytes and midbrain organoids, along with a reduced excitatory amino acid transporter 2 (EAAT2), the astrocytic glutamate transporter, expression. Conclusion: Our study was able to confirm in human-derived cellular models the previously demonstrated link between Park2 mutations and KAR upregulation [1]. The increased neuronal activity found in PARK2 midbrain organoids may be the expression of glutamate dysregulation. Two innovative findings emerged from this study: an increased TH expression in PARK2, matching the altered dopamine turnover related to such mutations and the contribution of astrocytes in Park2-associated excitotoxicity and neurodegeneration through an altered glutamate turnover. Further studies are required to establish Park2 role in TH expression and regulation, in astrocytic reactivity induction and in glutamate toxicity. References: [1] Maraschi A., Ciammola A., Folci A. et al. Parkin regulates kainate receptors by interacting with the GluK2 subunit. Nat Commun. (2014): 5:5182. [2] Zhang P., Xia N., Reijo Pera RA. Directed dopaminergic neuron differentiation from human pluripotent stem cells. J Vis Exp. (2014): (91):51737. [3] Santos R., Vadodaria KC., Jaeger BN. et al. Differentiation of Inflammation-Responsive Astrocytes from Glial Progenitors Generated from Human Induced Pluripotent Stem Cells. Stem Cell Reports. (2017): 1757-1769. [4] Lancaster MA., Knoblich JA. Generation of cerebral organoids from human pluripotent stem cells. Nat Protoc. (2014): 2329-40.