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Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

ABSTRACT: Here we describe the use of an organotypic hippocampal slice model for studying ?-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed ?-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous ?-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human ?-synuclein (S129G) using an adeno-associated viral (AAV) vector in ?-synuclein knockout slices. The processes of aggregation and spreading of ?-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of ?-synuclein aggregates and their prion-like spreading.

SUBMITTER: Elfarrash S

PROVIDER: S-EPMC6924077 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

Elfarrash Sara S Jensen Nanna Møller NM Ferreira Nelson N Betzer Cristine C Thevathasan Jervis Vermal JV Diekmann Robin R Adel Mohamed M Omar Nisreen Mansour NM Boraie Mohamed Z MZ Gad Sabry S Ries Jonas J Kirik Deniz D Nabavi Sadegh S Jensen Poul Henning PH

Acta neuropathologica communications 20191219 1

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disea ...[more]

PMID: 31856920

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Project description:The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deNEDDylase controlling ubiquitination activity of cullin-RING-E3 ligases (CRLs) and thus the levels of key cellular proteins. While the CSN and its catalytic subunit CSN5 have been extensively studied in cancer, its role in inflammatory and neurological diseases is less understood. Following verification that CSN5 is expressed in mouse and human brain, here we studied the role of the CSN in neuroinflammation and ischemic neuronal damage employing models of relevant brain-resident cell types, an ex vivo organotypic brain slice culture model, and the CRL NEDDylation state-modifying drugs MLN4924 and CSN5i-3, which mimic and inhibit, respectively, CSN5 deNEDDylase activity. Untargeted mass spectrometry-based proteomics revealed that MLN4924 and CSN5i-3 substantially alter the microglial proteome, including inflammation-related proteins. Applying these drugs and mimicking microglial and endothelial inflammation as well as ischemic neuronal stress by TNF and oxygen-glucose-deprivation/reoxygenation (OGD/RO) treatment, respectively, we could link CSN5/CSN-mediated cullin deNEDDylation to reduction of microglial inflammation, attenuated cerebral endothelial inflammation, improved barrier integrity, as well as protection from ischemic stress-induced neuronal cell death. Specifically, MLN4924 reduced phagocytic activity, motility, and inflammatory cytokine expression of microglial cells, and this was linked to inhibition of inflammation-induced NF-κB and Akt signaling. Inversely, Csn5 knockdown and CSN5i-3 increased NF-κB signaling. Moreover, MLN4924 abrogated TNF-induced NF-κB signaling in cerebral microvascular endothelial cells (hCMECs) and rescued hCMEC monolayers from OGD/RO-triggered barrier leakage, while CSN5i-3 exacerbated permeability. In an ex vivo organotypic brain slice model of ischemia/reperfusion stress, MLN4924 protected from neuronal death, while CSN5i-3 impaired neuronal survival. Neuronal damage was attributable to microglial activation and inflammatory cytokines, as indicated by microglial shape tracking and TNF-blocking experiments. Our results indicate a protective role of the CSN in neuroinflammation via brain-resident cell types involved in ischemic brain disease and implicate CSN activity-mimicking deNEDDylating drugs as potential therapeutics.

Dataset Information

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

Publications

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

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