Abstract:
Synucleinopathies are neurodegenerative diseases characterised by the abnormal accumulation of α-synuclein (αS) in perikarya and processes of neurons, so-called Lewy bodies (LB) and Lewy neurites (LN), respectively. Increasing evidence suggests a prion-like mechanism in the formation and propagation of αS lesions throughout the brain. Introduction of αS seeds induces progressive pathology in a variety of experimental models and in vitro cell-to-cell spreading of αS has been demonstrated between neurons. The aim of the present work was to study the formation, propagation, and cell-to-cell spread of αS lesions in an environment that mimics the brain environment. For this purpose, a hippocampal slice culture (HSC) model was established in which the hallmarks of α-synucleinopathies can be induced by a single treatment with αS seeds. LB- and LN-like hyperphosphorylated αS inclusions appear in a time- and concentration-dependent manner upon seed application and is followed by spreading of misfolded αS between different hippocampal regions. αS lesion-associated neurodegeneration could be monitored by neurofilament light (NfL) release into the culture medium. Potential therapeutic interference was demonstrated with an antibody directed against oligomeric and fibrillar αS, which attenuated seeding, seed-induced neurodegeneration, and spreading throughout the culture. The potential of this ex vivo brain culture system was then extended to adult human resection-derived brain cultures in which the induction of αS inclusions was also achieved. Rather surprisingly, in addition to the neuronal αS pathology, abundant inclusions were found in microglia of the αS lesion-bearing brain cultures. Similar microglial inclusions were also detected in adult αS transgenic mouse models. These microglial inclusions were immunoreactive with N-terminal αS antibodies but reveal a different conformation compared to the neuronal αS inclusions, as shown by conformation sensitive amyloid dyes. Microglial inclusions occur with some delay after the neuronal inclusions but were also found at presymptomatic stages in transgenic mice, suggesting a potential involvement of microglial αS in the progression of α-synucleinopathies. To investigate this further, microglia were depleted in brain cultures at different timepoints in respect to seed application. Results suggest that microglial inclusions develop secondarily to neuronal inclusions, and moreover imply a role of microglia in the spreading of neuronal inclusions. In summary, an ex vivo brain slice culture model system was established to study the induction and propagation of α-synucleinopathies. The importance of using a brain-like environment was exemplified by the discovery of an unexpected role of microglia in this process. The successful translation of the model system to human adult brain tissue will now allow to study mechanisms and therapeutic options of α-synucleinopathies in a true human adult brain environment.
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