A minimalistic co-culture platform for alpha-synuclein spreading in human dopaminergic neurons

Abstract

Parkinson’s disease (PD) is the second major neurodegenerative disease and the most common movement disorder. Due to age being a critical risk factor, the rapid ageing of the world population further increases the prevalence of PD. So far no treatment is available and therapies mainly focus on motor symptoms by pharmacologically substituting striatal dopamine, caused by the loss of dopaminergic neurons in the substantia nigra. This neuronal loss and intracellular protein aggregates, termed Lewy bodies (LBs), are pathological characteristics of PD. With disease progression, a spread of LBs through the brain can be observed which mainly follows axonal projections. Understanding the mechanisms of this progressive spread could be central to discovering the underlying molecular pathogenesis of the disease. As LBs mainly consist of alpha-synuclein (-syn), a prion-like spreading of -syn was suggested and is now widely accepted as a component in the PD pathogenesis. New dopaminergic model systems to study the exact mechanisms underlying -syn spread are urgently needed. As PD is a human disease, in vitro models should be derived from humans. Lund human mesencephalic (LUHMES) cells are a suitable alternative to other, mostly non-human, dopaminergic cell lines. However, difficulties cultivating them in microfluidics devices has made them thus far inaccessible for co-cultivation studies in the field of PD spreading. In the first part of this thesis, a human dopaminergic cell model system for studying the spreading of -syn fibrils is presented. First, the well-characterized LUHMES cell line was tested for suitability of PD research on prion-like spreading, as no data is currently available on this matter. For the analysis, immunofluorescence light microscopy was employed. An extended period of differentiation aimed for a high degree of neuronal maturity and long neurites to facilitate the connectivity of spatially-separated cell populations. Seeding experiments with -syn fibrils revealed a weak toxicity against these assemblies, even at prolonged differentiation. Second, to study the transmission of -syn fibrils via neuronal projections, we developed a light microscopy-compatible microfluidic co-culturing device, to maintain two LUHMES cell populations in separate cell compartments for up to two weeks of differentiation. During this time, a neurite network is formed which connects the fluidically isolated cell growth compartments. The ability to cultivate cells with neurites and soma in an isolated environment enabled seeding and transmission experiments in anterograde and retrograde directions. In the second part of this thesis, implementation strategies of the microfluidic co-culturing chip for alternative analysis methods are discussed. Firstly, the accessibility of the cells in the co-culturing device using a single-cell lysis instrument is evaluated. The tool allows for targeted lysis of individual adherent cells. Preliminary tests point in a promising direction, while LUHMES single cell lysate was successfully transferred to different analysis techniques. However, direct access to the channels of the microfluidic co-culturing chip was problematic and needs further modifications. Secondly, an implementation of the microfluidic device aiming for co-cultivation of LUHMES cells on electron microscopy grids to study neurite architecture was pursued. Thereby, microfluidic devices harbor only cell soma, but neurites can grow onto an electron microscopy grid, as only they are thin enough to be visualized by cryo-electron microscopy. Proof-of-concept experiments demonstrate the direct visualization of LUHMES cell neurites in a near-native, frozen-hydrated state.