INVESTIGATING GLUTAMATE TOXICITY ASSOCIATED TO PARK2 MUTATIONS IN PRE-CLINICAL MODELS OF PARKINSON¿S DISEASE.

ABSTRACT Background: Park2 mutations cause Autosomal Recessive Juvenile Parkinsonism (ARJP), characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Park2 encodes for a ubiquitin-ligase protein whose mutation upregulates Gluk2, a subunit of the glutamate kainate receptor (KAR), expressed in SNpc neurons. Park2 is highly expressed also in astrocytes and KARs upregulation may induce excitotoxicity both in DA neurons and glia, leading to an early synaptopathy, neuroinflamation and neurodegeneration. Aims and Objectives: 1. To generate Park2 induced pluripotent stem cells (iPSCs)-derived in vitro cellular models; 2. To characterize Park2 iPSCs-derived in vitro cellular models; 3. to test glutamate toxicity due to KAR upregulation in Park2 cellular models. Materials and Methods: Fibroblasts and lymphocytes from Park2 patients and age-matched controls were reprogrammed into iPSCs. The iPSCs were further differentiated into dopaminergic neurons, astrocytes and mesencephalic organoids were generated and differentiated. 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: Gluk2 levels resulted significantly increased in PARK2 midbrain organoids compared to CTR both at WB (p< 0.001) and qPCR analyses (p< 0.001). Gluk2 levels resulted also significantly enhanced in PARK2 astrocytes both at WB (p< 0.05) and qPCR analyses (p< 0.05). TH mRNA and protein levels were significantly increased both in PARK2 dopaminergic neurons (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) and midbrain organoids (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) compared to CTR. Glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, resulted enhanced in PARK2 astrocytes and especially in PARK2 midbrain organoids (WB p< 0.001; IF p< 0.01). EAAT2, the astrocytic glutamate transporter resulted reduced in mutated lines (WB p< 0.01). Calcium-imaging and HD-MEAs show an oscillatory augmented reactivity in PARK2 midbrain organoids. Conclusions and perspectives: Gluk2 expression was enhanced in PARK2 astrocytes and midbrain organoids, confirming the previous finding that Park2 mutations lead to KAR upregulation (Maraschi A, 2014). Neuronal reactivity was also found increased in PARK2 midbrain organoids at electrophysiology assessment, maybe linked to glutamate dysregulation. Two innovative findings emerged from this study. First of all, that TH expression resulted increased in PARK2, supporting previous finding that stated an augmented dopamine turnover and a reduced dopamine re-uptake (Jiang H., 2012). This is an impairment that happens early in the neurodegenerative process and that could consequently lead to an excessive oxidative stress and consequent neurodegeneration. The second original result is that PARK2 is associated to an increased astrocytic reactivity and a possible dysfunction of astrocytic glutamate transporter EAAT2. This finding means that astrocytes play a key role in neurodegeneration although it is not clear whether they contribute to the initiation or propagation of it. Their increased reactivity could be the consequence of a glutamate toxicity or glutamate toxicity could result from reactive astrocytes dysfunction, not able to process the excessive glutamate influx. Further studies are required in order to establish Park2 role in TH expression regulation, in astrocytic reactivity induction and in glutamate toxicity.