Abstract :
[en] Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons
exhibiting Lewy Pathology (LP) in the substantia nigra. It has been suggested that LP
to occurs at the gastrointestinal tract long before than in the brain. Braak’s hypothesis
postulates that external agents in the gut come in contact with enteric neurons
triggering α-synuclein misfolding and aggregation, which then propagates through
the vagal nerve towards the brain. Numerous preclinical and clinical studies support
this hypothesis; however, it is yet impossible to observe this process in a human in
vivo. To overcome this, this project proposed the construction of an in vitro
assembloid model of the intestine and the enteric nervous system based on iPSCderived
organoids. The assembloid, composed of intestinal organoids (iHOs) and
enteric neurons (ENS), was used to visualize the pathological spread of α-synuclein.
Two different approaches were followed to build the assembloids. The first one
involved the co-cultivation of the iHOs and ENS in a single well subdivided by an
insert (iHO-ENS assembloid). Intracellular and extracellular α-synuclein were
quantified using blotting techniques. The second strategy involved combining both
cell types at earlier differentiation stages and culturing them in 3D conditions to
obtain iHOs with integrated ENS (iHO-ENC assembloid). In this case, α-synuclein
was measured using high content microscopy and an own-developed computational
algorithm. This algorithm was first created for the analysis of brain organoids and
later adapted to the assembloids. In the iHO-ENS assembloid, direct contact within
the different cell types could not be detected, and even though extracellular α-
synuclein measurements suggested that iHO-secreted α-synuclein might be up taken
by the ENS, this could not be confirmed by intracellular α-synuclein tests. By
contrast, an increased content of α-synuclein was detected in neurons co-cultured
with PD iHOs in the iHO-ENC assembloid. Furthermore, aggregate size within the
neurons conveyed early stages of LP. Interestingly, in the iHOs α-synuclein
expression was detected mostly within enteroendocrine cells, which have been
proposed as α-synuclein misfolding induction sites in the gut. Additionally, analysis of
PD brain organoids revealed an increased content of α-synuclein phosphorylated at
S129 in nuclei, supporting the function of this modification as a nuclear localization
tag, and providing insight into its pathological implications. Furthermore, in this model
Lewy body-like aggregates expanded over time mimicking pathological progression.
Overall, findings of this project encompass the novel characterization of α-synuclein
expression in iHOs and, preliminarily, the foremost observation of α-synuclein
propagation from the intestine to the enteric nervous system in a human organoidbased
model. Both the cellular and computational models created can serve as the
foundation for further disease modeling and drug screening studies.
