Accelerated dopaminergic differentiation precedes neuronal loss in a 3xSNCA midbrain organoid
model of Parkinson’s disease

Parkinson’s disease (PD) is a complex, progressive neurodegenerative disease characterized by the
loss of dopaminergic neurons in the substantia nigra pars compacta. While it is well established that
by the time PD patients are diagnosed, they have lost between 40-60% of their dopaminergic
neurons in the substantia nigra, the molecular and cellular events that precede this neuronal decline
are poorly understood. Here we use patient specific human midbrain organoids to investigate the
molecular and cellular changes that precede neurodegeneration in PD. Midbrain organoids are
generated from three PD patient specific induced pluripotent stem cell lines with the SNCA
triplication mutation, along with two sex-age matched healthy control lines and two isogenic controls. We investigate differences between wild type and SNCA mutant organoids with respect to
neural stem cell activity as well as neuronal, synaptic and astrocytic development at 15, 21, 30, 35, 70
and 90 days of organoid maturation. This is done by assessing the expression of relevant key markers
using immunofluorescence stainings, Western blots and other biochemical assays. Our results show
that organoids derived from PD patients with the SNCA triplication mutation show increased stem
cell activity and accelerated neuronal differentiation in comparison to healthy controls at early time
points of organoid maturation. The accelerated neuronal differentiation in the mutants is followed by
the loss of dopaminergic neurons at later time points. Over the time course, we also observe nuclear
laminar deficits in the cells within the mutant organoids and the upregulation of senescence-
associated markers. Our results suggest that SNCA triplication organoids show an atypical
development that may increase the vulnerability of dopaminergic neurons to degeneration through
accelerated aging.
Keywords: Parkinson’s disease, α-synuclein, neurodegeneration, neuroplasticity

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