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Extracellular vesicles from amyloid-? exposed cell cultures induce severe dysfunction in cortical neurons.

ABSTRACT: Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-? (A?) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of A?42 protofibrils by astrocytes results in the release of EVs containing neurotoxic A?. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and A?42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from A?42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from A? exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of A?-induced pathology.

SUBMITTER: Beretta C

PROVIDER: S-EPMC7661699 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons.

Beretta Chiara C Nikitidou Elisabeth E Streubel-Gallasch Linn L Ingelsson Martin M Sehlin Dag D Erlandsson Anna A

Scientific reports 20201112 1

Alzheimer's disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ<sub>42</sub> protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cel ...[more]

PMID: 33184307

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Project description:Stem-cell-derived extracellular vesicles (EVs) are promising tools for therapeutic delivery and imaging in the medical research fields. EVs that arise from endosomal compartments or plasma membrane budding consist of exosomes and microvesicles, which range between 30 and 200 nm and 100-1000 nm, respectively. Iron oxide nanoparticles can be used to label stem cells or possibly EVs for magnetic resonance imaging. This could be a novel way to visualize areas in the body that are affected by neurological disorders such as stroke. Human induced pluripotent stem cells (iPSK3 cells) were plated on low-attachment plates and treated with SB431542 and LDN193189 during the first week for the induction of cortical spheroid formation and grown with fibroblast growth factor 2 and cyclopamine during the second week for the neural progenitor cell (iNPC) differentiation. iNPCs were then grown on attachment plates and treated with iron oxide (Fe3O4) nanoparticles at different sizes (8, 15, and 30 nm in diameter) and concentrations (0.1, 10, and 100 μM). The spheroids and media collected from these cultures were used for iron oxide detection as well as EV isolation and characterizations, respectively. MTT assay demonstrated that the increased size and concentration of the iron oxide nanoparticles had little effect on the metabolic activity of iNPCs. In addition, the Live/Dead assay showed high viability in all the nanoparticle treated groups and the untreated control. The EVs isolated from these culture groups were analyzed and displayed similar or higher EV counts compared with control. The observed EV size averaged 200-250 nm, and electron microscopy revealed the expected exosome morphology for EVs from all groups. RT-PCR analysis of EV biogenesis markers (CD63, CD81, Alix, TSG101, Syntenin1, ADAM10, RAB27b, and Syndecan) showed differential expression between the iron-oxide-treated cultures and nontreated cultures, as well as between adherent and nonadherent 3D cultures. Iron oxide nanoparticles were detected inside the cortical spheroid cells but not EVs by MRI. The addition of iron oxide nanoparticles does not induce significant cytotoxic effects to cortical spheroids. In addition,, nanoparticles may stimulate the biogenesis of EVs when added to cortical spheroids in vitro.

Project description:Neurons derived from human induced pluripotent stem cells (hiPSCs) are powerful tools for modeling neural pathophysiology and preclinical efficacy/toxicity screening of novel therapeutic compounds. However, human neurons cultured in vitro typically do not fully recapitulate the physiology of the human nervous system, especially in terms of exhibiting morphological maturation, longevity, and electrochemical signaling ability comparable to that of adult human neurons. In this study, we investigated the potential for astrocyte-derived extracellular vesicles (EVs) to modulate survival and electrophysiological function of human neurons in vitro. Specifically, we demonstrate that EVs obtained from human astrocytes promote enhanced single cell electrophysiological function and anti-apoptotic behavior in a homogeneous population of human iPSC-derived cortical neurons. Furthermore, EV-proteomic analysis was performed to identify cargo proteins with the potential to promote the physiological enhancement observed. EV cargos were found to include neuroprotective proteins such as heat shock proteins, alpha-synuclein, and lipoprotein receptor-related protein 1 (LRP1), as well as apolipoprotein E (APOE), which negatively regulates neuronal apoptosis, and a peroxidasin homolog that supports neuronal oxidative stress management. Proteins that positively regulate neuronal excitability and synaptic development were also detected, such as potassium channel tetramerization domain containing 12 (KCTD12), glucose-6- phosphate dehydrogenase (G6PD), kinesin family member 5B (KIF5B), spectrin-alpha non-erythrocytic1 (SPTAN1). The remarkable improvements in electrophysiological function and evident inhibition of apoptotic signaling in cultured neurons exposed to these cargos may hold significance for improving preclinical in vitro screening modalities. In addition, our collected data highlight the potential for EV-based therapeutics as a potential class of future clinical treatment for tackling inveterate central and peripheral neuropathies.

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Extracellular vesicles from amyloid-? exposed cell cultures induce severe dysfunction in cortical neurons.

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Extracellular vesicles from amyloid-β exposed cell cultures induce severe dysfunction in cortical neurons.

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