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Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain.

ABSTRACT: Increased production, oligomerization and aggregation of amyloid-? (A?) peptides are hallmark pathologies of Alzheimer's disease (AD). Expressing familial AD mutations (amyloid precursor protein and/or presenilins mutations), the A?-pathologies of AD has been recapitulated in animal models of AD. Very few primary cell culture-based models of AD are available and they exhibit very weak A?-pathologies compared to what is seen in AD patients and animal models of AD. CNS stem/progenitor cells are present in both embryonic and adult brains. They can be isolated, grown as neurospheres and differentiated into neurons, astrocytes and oligodendrocytes. It is not yet known whether CNS stem/progenitor cells can support the production of A? peptides in culture. In this report, we have established A?-pathologies such as production, secretion, oligomerization and aggregation of A? peptides utilizing neurosphere cultures to create a new cellular model of AD. These cultures were developed from E15 embryonic brains of transgenic mice carrying the Swedish mutations in humanized mouse APP cDNA and the exon-9 deleted human presenilin 1 cDNA both regulated by mouse prion protein gene (Prnp) promoter. Results demonstrated the expression of transgene transcripts, APPswe protein and its processed products only in transgene positive neurosphere cultures. These cultures generate and secrete both A?40 and A?42 peptides into culture medium at levels comparable to the A? load in the brain of AD patients and animal models of AD, and produce pathogenic oligomers of A? peptides. The A?42/A?40 ratio in the medium of transgene positive neurosphere cultures is higher than any known cellular models of AD. Conformation dependent immunocytochemistry demonstrated the possible presence of intracellular and extracellular aggregation of A? peptides in neurosphere cultures, which are also seen in AD brain and animal models of AD. Collectively, our neurosphere cultures provide robust A?-pathologies of AD better than existing cellular model of Alzheimer's disease.

SUBMITTER: Ghate PS

PROVIDER: S-EPMC4137416 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain.

Ghate Pankaj S PS Sidhar Himakshi H Carlson George A GA Giri Ranjit K RK

SpringerPlus 20140326

Increased production, oligomerization and aggregation of amyloid-β (Aβ) peptides are hallmark pathologies of Alzheimer's disease (AD). Expressing familial AD mutations (amyloid precursor protein and/or presenilins mutations), the Aβ-pathologies of AD has been recapitulated in animal models of AD. Very few primary cell culture-based models of AD are available and they exhibit very weak Aβ-pathologies compared to what is seen in AD patients and animal models of AD. CNS stem/progenitor cells are pr ...[more]

PMID: 25140287

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Project description:BackgroundAmyloid-β deposition and accumulation of autophagic vacuoles are pathologic features of Alzheimer's disease (AD). Dysregulation of the endosomal-autophagic-lysosomal (EAL) pathway, which impairs amyloid precursor protein processing, is one of the earliest changes in AD. However, the precise role of EAL pathway in neurodegeneration remains unclear. This study aimed to investigate the role of EAL pathway in AD and further study the mechanism of EAL dysfunction.MethodsWe used 3-, 7-, and 12-month-old APPswe/PSEN1dE9 (APP/PS1) mice to model different stages of AD with age- and gender-matched wild-type littermates as controls (4-7 mice per group) and detected the changes of EAL markers, endosomal organizers Rab5 and Rab7, autophagosome marker LC3B, and lysosomal proteins Lamp1/2 in cortex and hippocampus by immunohistochemistry and Western blotting analysis. To further explore the mechanism of EAL dysregulation in AD, components of the class III phosphatidylinositol 3-kinase (PI3KC3) complex, activators of Rab7 (Beclin1 and UVRAG), and the negative regulator of Rab7 (Rubicon) were also measured in this two brain regions.ResultsIn 7-month-old APP/PS1 brain that amyloid beta initiated to accumulate intracellularly, EAL pathway, and related PI3KC3 members, UVRAG and Beclin1 were upregulated both in cortex and hippocampus (all P < 0.05). By the age of 12 months old, when abundant amyloid plaques formed, EAL markers, UVRAG, and Beclin1 were also upregulated in the cortex (all P < 0.05). However, Rab7 was decreased significantly (P = 0.0447), accompanied by a reduction of its activating PI3KC complex component Beclin1 (P = 0.0215) and enhancement of its inhibiting component Rubicon (P = 0.0055) in the hippocampus.ConclusionsOur study implies that EAL pathway, represented as Rab7 and its PI3KC3 regulators' expressions, showed temporal and spatial variation in brains at different stages of AD. It provides new insights into the role of EAL pathway in pathogenesis and indicates potential therapeutic targets in neurodegenerative diseases.

Dataset Information

Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain.

Publications

Development of a novel cellular model of Alzheimer's disease utilizing neurosphere cultures derived from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J embryonic mouse brain.

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