Abstract
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 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 Aβ peptides in culture. In this report, we have established Aβ-pathologies such as production, secretion, oligomerization and aggregation of Aβ 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 Aβ40 and Aβ42 peptides into culture medium at levels comparable to the Aβ load in the brain of AD patients and animal models of AD, and produce pathogenic oligomers of Aβ peptides. The Aβ42/Aβ40 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 Aβ peptides in neurosphere cultures, which are also seen in AD brain and animal models of AD. Collectively, our neurosphere cultures provide robust Aβ-pathologies of AD better than existing cellular model of Alzheimer’s disease.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-161) contains supplementary material, which is available to authorized users.
Highlights
Alzheimer’s disease (AD) is a chronic, irreversible and progressive neurodegenerative disease
Development of Central nervous system (CNS) stem/progenitor cells cultures from B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J mice Embryonic day 15 (E15) brain cells isolated from B6C3Tg(APPswe,PSEN1dE9)85Dbo/J mice grew as balls of cells termed as neurospheres (NS) within 4-5 days in culture (Figure 1A)
polymerase chain reaction (PCR) assay for huAPPswe and huPSEN1dE9 transgenes demonstrated the amplification of a 350-bp and a 608-bp DNA fragments respectively from NS1, NS3 and a Tg+ve mouse genomic DNA but not from NS2 and NS4 lines (Figure 1B) showing that, NS1 and NS3 lines are Tg+ve, whereas NS2 and NS4 are Tg-ve
Summary
Alzheimer’s disease (AD) is a chronic, irreversible and progressive neurodegenerative disease. AD is characterized by extracellular deposition of amyloid-β (Aβ) peptides as senile plaques (Glenner and Wong 1984), intraneuronal neurofibrillary tangles (NFT) (Kosik et al 1986; Wood et al 1986), astrogliosis (Rodriguez et al 2009), microglial activation (Giri et al 2003) and loss of synapses and neurons in in the cascade that eventually leads to AD associated neurodegeneration (Hardy and Allsop 1991; Sommer 2002) Utilizing this hypothesis, transgenic animals were created using human genes linked to FAD such as APP and PSEN1. Quicker, cheaper and reproducible alternative models were explored utilizing cell culture based systems
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