The iPSC‐derived human dopamine neurons from a healthy subject and a patient with α‐synuclein triplication reveal novel therapeutic target

Parkinson's disease (PD) is characterized by progressive loss of dopamine (DA) neurons in the substantia nigra (SN‐A9). α‐synuclein has been genetically implicated in familial and sporadic PD and is associated with PD susceptibility, progression and pathology. A cardinal feature of neurons that separates them from nearly all other cell types is their excitability. Multiple channels and transporters regulate the excitability of DA neurons. DA neurons are uniquely defined by expression of Dopamine Transporter (DAT), D2 autoreceptor and GIRK, where they govern the excitability of DA neurons, and thus DA neurotransmission. To determine the impact of α‐synuclein on the activity of DA neurons, we differentiated induced pluripotent stem cells (iPSC) from PD patients with α‐synuclein triplication and the healthy sibling. Two sets of iPSCs used in our studies: iPSCs with triplication of α‐synuclein (AST), and iPSCs with normal α‐synuclein (NAS) levels. The A9 human‐like DA neurons were differentiated and identified by expression of Tyrosine Hydroxylase (TH), GIRK2, VMAT2, DAT, Nurr1, FOXA2, as phenotypic markers for DA neurons. The expression of α‐synuclein and Parkin, two PD‐associated proteins were higher in the AST‐derived human‐like DA neurons as compared to the NAS‐derived human‐like DA neurons. We found significant morphological differences between neurons with normal α‐synuclein levels (NAS) vs. neurons with α‐synuclein triplication (AST). The AST neurons exhibited significantly larger soma, short neurites and lower D2R and GIRK expression ( P <0.005). Importantly, our differentiation protocol resulted in spontaneously firing human‐like DA neurons. Whole‐cell voltage‐clamp recordings revealed no difference in the overall activity of sodium and potassium channels in the AST and NAS DA neurons (( P >0.05). Consistent with our previous report in mice DA neurons (Lin et al., 2016), the majority of NAS‐derived DA neurons exhibited a mixture of single spikes and small burst activity with an underlying “pacemaker‐like” periodicity. Whereas, ~90% of AST‐derived DA neurons exhibited a unique firing pattern of spontaneous firing activity with a pause between subsequent broadbrimmed burst clusters on a high depolarized plateau (up state) ( P <0.05). We found, in the AST‐derived DA neurons, activation of D2R or GIRK channels reduced the amplitude and the width of the up state and suppressed the firing frequency, suggesting α‐synuclein triplication disrupts firing frequency of DA neurons and thus DA transmission via dysregulation of D2R and GIRK channels.Morphological differences and their measurements after 4 months differentiation of NAS and AST dopamine neuronsOur differentiation protocol produced spontaneously active DA neurons. Increased α‐Syn dysregulated the spontaneous firing activity of AST DA neuronsQuinpirole activation of D2R restores endogenous AST‐derived human‐like DA neuronsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .