gms | German Medical Science

Objective: Since the 1960s, there has been some success in targeted medical (L-Dopa) and surgical (deep brain stimulation) treatments for motor symptoms in Parkinson’s disease (PD). However, these are non-disease modifying, each with limitations. An experimental and emerging therapy for PD involves stereotactic dopaminergic cell transplantation. Clinical trials in the 1990s were proof of concept as marked improvement was seen in some patients receiving fetal grafts into striatum. Although, on autopsy some grafts acquired Lewy body pathology, a hallmark of PD. This host-to-graft spread of disease is potentially a major hindrance for future treatments. We aim to develop an in vitro model of disease spread using transgenic cell lines over-expressing α-synuclein, the protein responsible for Lewy body pathology, for mechanistic insight into this process.

Methods: A human embryonic stem cell (hESC) line was transfected with an expression plasmid containing the human SNCA gene to produce several clonal transgenic lines over-expressing α-synuclein. Of these, the S37 line expressed eight-fold greater levels of α-synuclein than controls. The transgenic S37 and control hESCs were differentiated into midbrain dopaminergic neurons using a robust protocol, and culture media was seeded with recombinant monomers and pre-formed fibrils (PFFs) of α-synuclein. After 3 weeks, the neurons were fixed and immunofluorescence used to detect expression of phosphorylated α-synuclein at serine-129 (pSer-129), an early sign of Lewy pathology.

Results: Monomers of α-synuclein did not cause accumulation of phosphorylated α-synuclein in either S37 or control neurons. PFFs did cause the formation of phosphorylated α-synuclein structures in both sets of neurons. The pSer-129 α-synuclein immunostaining was observed in discrete short stretches within axons, an early sign of Lewy neurite formation. This preliminary work shows that the phosphorylated α-synuclein structures was more prevalent in S37 neurons, which highly express α-synuclein, than in control neurons.

Conclusion: We found that monomers of α-synuclein cannot seed Lewy body-like pathology in human dopaminergic neurons agreeing with work performed with mouse and rat neurons. Furthermore, α-synuclein PFFs can initiate signs of Lewy pathology (pSer-129 α-synuclein in axons) in both wild-type and S37 transgenic neurons, with the latter accumulating more pathology. This work establishes an important human model to study PD within neuronal networks where neurons that highly express endogenous α-synuclein may be more susceptible to Lewy pathology and disease. Further work will be done on S37 and other transgenic SNCA lines expressing varying α-synuclein levels to quantitatively correlate differences in rate of disease spread to the levels of α-synuclein protein expression. This work will be subsequently validated in a rat model of PD in vivo.