Network hyperexcitability and dopaminergic neuron loss in a novel A30P-driven Parkinson’s midbrain organoid model
by Kea Aline Schmoll
Date of Examination:2025-04-10
Date of issue:2025-11-24
Advisor:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:Dr. Hauke Werner
Referee:Prof. Dr. Kristina Lorenz
Persistent Address:
http://dx.doi.org/10.53846/goediss-11634
Files in this item
Name:Thesis_KS_final.pdf
Size:4.69Mb
Format:PDF
Name:Schmoll_revisionsschein ausgefüllt.pdf
Size:327.Kb
Format:PDF
This file will be freely accessible after 2026-04-09.
Abstract
English
Midbrain dopaminergic (mDA) neurons are essential for motor control, reward processing, emotional regulation and motivation. The degeneration of these neurons, seen in Parkinson’s disease (PD), disrupts fine motor control, leading to symptoms such as bradykinesia, rigidity, and tremor. To date, there is no cure for Parkinson’s Disease (PD), and the mechanisms underlying dopaminergic neuron degeneration remain elusive. Current cell therapies focus on replenishing the degenerated dopaminergic neurons, by transplantation of dopaminergic neurons derived from human pluripotent stem cells (PSCs). Nevertheless, these therapies face post-transplantation challenges such as low cell survival, consistent dopamine release, and co-development of an undesired cell population such as serotonergic neurons leading to hyperkinesia. Midbrain organoids generated from PD hiPSCs offer a promising platform to investigate PD pathomechanisms and discover novel therapeutic approaches. This study aimed to establish a PD model in a novel midbrain organoid and employ optogenetics to control mDA neuron activity and thus dopamine release. We developed a robust mBENO protocol featuring bona fide midbrain dopaminergic neurons within a functional neuronal-glial network resembling the human midbrain/basal ganglia. The composition of mBENO was characterized by temporal transcriptomic analysis and morphological analyses after immunofluorescence labelling, while its function was assessed through local field potential recordings and dopamine content measurements. Neurotransmitter administration confirmed the presence of a functional neuronal network harboring dopaminergic, GABAergic, cholinergic and glutamatergic neurons. Next, we genome engineered and established a PD mBENO model by introducing a SNP found in PD patients, the α-synuclein (SNCA) A30P mutation. SNCA A30P did not impair the differentiation potential but led to dopaminergic neuron loss by D90 and electrophysiological hyperexcitability resembling basal ganglia dysfunction in PD patients. This SNCA A30P mBENO model provides a valuable system to investigate PD pathomechanisms, disease progression, as well as potential therapeutic targets. mBENO could potentially serve as a cell source for cell replacement therapies, offering better cell survival and retention in patients compared to singularized cell injections. Furthermore, to control dopamine secretion we engineered an optogenetic cell line, SynfChrimson, which expresses a stably integrated, fast, red light activatable channel (f-Chrimson), under the control of a synapsin promotor in the AAVS1 locus. Multielectrode array analysis confirmed that the SynfChrimson cell line exhibits neuron specific light-evoked activation. Implanting optogenetically controlled mDA neurons along with a medical device could potentially overcome the challenges of fine-tuning dopamine release in PD therapy. In conclusion, we present a novel midbrain organoid model, which recapitulates the human midbrain in cellular composition and function. We demonstrate that the A30P mBENO exhibits mDA neuron loss and hyperexcitability, functionally modeling PD. The A30P mBENO holds promise as a tool to investigate pathomechanisms and pharmaceutical targets in the future.
Keywords: midbrain organoid; Parkinson's Disease; hiPSCs; mBENO; optogenetics; fChrimson; A30P
