Abstract
BackgroundProteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression.MethodsAlpha-synuclein (αS) inclusions were induced in long-term murine and human slice cultures by seeded aggregation. An αS seed-recognizing human antibody was tested for blocking seeding and/or spreading of the αS lesions. Release of neurofilament light chain (NfL) into the culture medium was assessed.ResultsTo study initial stages of α-synucleinopathies, we induced αS inclusions in murine hippocampal slice cultures by seeded aggregation. Induction of αS inclusions in neurons was apparent as early as 1week post-seeding, followed by the occurrence of microglial inclusions in vicinity of the neuronal lesions at 2–3 weeks. The amount of αS inclusions was dependent on the type of αS seed and on the culture’s genetic background (wildtype vs A53T-αS genotype). Formation of αS inclusions could be monitored by neurofilament light chain protein release into the culture medium, a fluid biomarker of neurodegeneration commonly used in clinical settings. Local microinjection of αS seeds resulted in spreading of αS inclusions to neuronally connected hippocampal subregions, and seeding and spreading could be inhibited by an αS seed-recognizing human antibody. We then applied parameters of the murine cultures to surgical resection-derived adult human long-term neocortical slice cultures from 22 to 61-year-old donors. Similarly, in these human slice cultures, proof-of-principle induction of αS lesions was achieved at 1week post-seeding in combination with viral A53T-αS expressions.ConclusionThe successful translation of these brain cultures from mouse to human with the first reported induction of human αS lesions in a true adult human brain environment underlines the potential of this model to study proteopathic lesions in intact mouse and now even aged human brain environments.
Highlights
Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms
Induction of αS inclusions in murine hippocampal slice cultures Murine hippocampal slice cultures (HSCs) were prepared from postnatal days 4–6 Thy1-h[A53T]αS tg mice overexpress human mutant (A53T)]αS tg or wt mice and grown in culture for 10 days to stabilize. αS inclusions were induced by onetime application of αS seed-rich aged Thy1-h[A53T]αS transgenic mouse brain homogenate or alternatively by synthetic αS preformed fibrils (Fig. 1A)
We further provided proof-of-principle evidence for their clinical application in screening antibodies that prevent the spread of αS lesions
Summary
Proteopathic brain lesions are a hallmark of many age-related neurodegenerative diseases including synucleinopathies and develop at least a decade before the onset of clinical symptoms. Prion-like transmission of αS lesions was proposed and the concept of seeded aggregation – a nucleation-dependent process similar to the one described for prions and amyloid-beta (Aβ) – was suggested [3, 4]. This concept gained further support by in vitro findings showing that the exogenous application of αS fibrils induced Lewy body-like pathology in cultured neurons [5,6,7,8]. While mice have been instrumental in the past to study such prion-like propagation of pathogenic seeds [16], mouse models are time-consuming and costly, and experimental manipulations to mechanistically understand propagation of seeds are challenging
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