Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures.

BACKGROUND: 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. Thus, understanding of the initiation and propagation of such lesions is key for developing therapeutics to delay or halt disease progression. METHODS: Alpha-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. RESULTS: To 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. CONCLUSION: The 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.

Conversion of Synthetic Aß to In Vivo Active Seeds and Amyloid Plaque Formation in a Hippocampal Slice Culture Model.

UNLABELLED: The aggregation of amyloid-ß peptide (Aß) in brain is an early event and hallmark of Alzheimer's disease (AD). We combined the advantages of in vitro and in vivo approaches to study cerebral ß-amyloidosis by establishing a long-term hippocampal slice culture (HSC) model. While no Aß deposition was noted in untreated HSCs of postnatal Aß precursor protein transgenic (APP tg) mice, Aß deposition emerged in HSCs when cultures were treated once with brain extract from aged APP tg mice and the culture medium was continuously supplemented with synthetic Aß. Seeded Aß deposition was also observed under the same conditions in HSCs derived from wild-type or App-null mice but in no comparable way when HSCs were fixed before cultivation. Both the nature of the brain extract and the synthetic Aß species determined the conformational characteristics of HSC Aß deposition. HSC Aß deposits induced a microglia response, spine loss, and neuritic dystrophy but no obvious neuron loss. Remarkably, in contrast to in vitro aggregated synthetic Aß, homogenates of Aß deposits containing HSCs induced cerebral ß-amyloidosis upon intracerebral inoculation into young APP tg mice. Our results demonstrate that a living cellular environment promotes the seeded conversion of synthetic Aß into a potent in vivo seeding-active form. SIGNIFICANCE STATEMENT: In this study, we report the seeded induction of Aß aggregation and deposition in long-term hippocampal slice cultures. Remarkably, we find that the biological activities of the largely synthetic Aß aggregates in the culture are very similar to those observed in vivo This observation is the first to show that potent in vivo seeding-active Aß aggregates can be obtained by seeded conversion of synthetic Aß in a living (wild-type) cellular environment.

Seeded strain-like transmission of ß-amyloid morphotypes in APP transgenic mice.

The polymorphic ß-amyloid lesions present in individuals with Alzheimer's disease are collectively known as cerebral ß-amyloidosis. Amyloid precursor protein (APP) transgenic mouse models similarly develop ß-amyloid depositions that differ in morphology, binding of amyloid conformation-sensitive dyes, and Aß40/Aß42 peptide ratio. To determine the nature of such ß-amyloid morphotypes, ß-amyloid-containing brain extracts from either aged APP23 brains or aged APPPS1 brains were intracerebrally injected into the hippocampus of young APP23 or APPPS1 transgenic mice. APPPS1 brain extract injected into young APP23 mice induced ß-amyloid deposition with the morphological, conformational, and Aß40/Aß42 ratio characteristics of ß-amyloid deposits in aged APPPS1 mice, whereas APP23 brain extract injected into young APP23 mice induced ß-amyloid deposits with the characteristics of ß-amyloid deposits in aged APP23 mice. Injecting the two extracts into the APPPS1 host revealed a similar difference between the induced ß-amyloid deposits, although less prominent, and the induced deposits were similar to the ß-amyloid deposits found in aged APPPS1 hosts. These results indicate that the molecular composition and conformation of aggregated Aß in APP transgenic mice can be maintained by seeded conversion.