Characterization of Prion Susceptibility in Neuro2a Mouse Neuroblastoma Cell Subclones

Uryu, Masahide; Karino, Ayako; Kamihara, Yukiko; Horiuchi, Motohiro

doi:10.1111/j.1348-0421.2007.tb03954.x

Cited by 23 publications

(37 citation statements)

References 43 publications

(55 reference statements)

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“…2). The CNs supported prion propagation with various prion strains including Obihiro strain, which is not propagated well in N2a-3 and GT1-7 cells (Uryu et al, 2007). Difference in PrP Sc stains in CNs infected with Obihiro strain (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…PK digestion was terminated by the addition of Pefabloc (Roche) to 1 mM, and the lysates were then treated with 50 µg DNase I ml À1 at room temperature for 15 min. Precipitation of PrP-res by phosphotungstic acid (PTA) followed by SDS-PAGE, Western transfer, and chemiluminescence detection were carried out as described elsewhere (Shindoh et al, 2009;Uryu et al, 2007). For detection of GFAP and b-III tubulin, the procedures from PK digestion to PTA precipitation were omitted, and 2 µg of total protein was loaded onto each lane.…”

Section: Methodsmentioning

confidence: 99%

“…ScN2a-3-22L (Nakamitsu et al, Immunoprecipitates from GT1-7 (GT1) or ScGT-17-22L (ScGT1) cells with mAb 8D5 or negative control mAb (NC) were subjected to immunoblotting using mAb 110, which recognizes the epitope in the octapeptide repeat at aa 59-65 and 83-89. 2010), ScN2a-3-Ch (Uryu et al, 2007) and GT1-7 persistently infected with the 22L prion strain (ScGT1-7-22L) (Yamasaki et al, 2012) were used as prion-infected cells. Those cells were cultured as described previously (Uryu et al, 2007;Yamasaki et al, 2012) and used at passage history between 5 and 30.…”

Section: Methodsmentioning

confidence: 99%

“…2010), ScN2a-3-Ch (Uryu et al, 2007) and GT1-7 persistently infected with the 22L prion strain (ScGT1-7-22L) (Yamasaki et al, 2012) were used as prion-infected cells. Those cells were cultured as described previously (Uryu et al, 2007;Yamasaki et al, 2012) and used at passage history between 5 and 30.…”

Section: Methodsmentioning

confidence: 99%

“…Subclones of the Neuro2a mouse neuroblastoma cell line, N2a-3 (Uryu et al, 2007), and the GT1 hypothalamic neuronal cell line, GT1-7 (Schatzl et al, 1997) were used. ScN2a-3-22L (Nakamitsu et al, Immunoprecipitates from GT1-7 (GT1) or ScGT-17-22L (ScGT1) cells with mAb 8D5 or negative control mAb (NC) were subjected to immunoblotting using mAb 110, which recognizes the epitope in the octapeptide repeat at aa 59-65 and 83-89.…”

Section: Methodsmentioning

confidence: 99%

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Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining

Tanaka

Fujiwara

Suzuki

et al. 2016

Journal of General Virology

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We established abnormal isoform of prion protein (PrP Sc )-specific double immunostaining using mAb 132, which recognizes aa 119-127 of the PrP molecule, and novel PrP Sc -specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrP Sc -specific double immunostaining, we analysed PrP Sc in immortalized neuronal cell lines and primary cerebralneuronal cultures infected with prions. The PrP Sc -specific double immunostaining showed the existence of PrP Sc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrP Sc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrP Sc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrP Sc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrP Sc stains were predominantly observed on the surface of the 22 L straininfected primary cerebral neurons. Only 14 % of PrP Sc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrP Sc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrP Sc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrP Sc that possesses the Nterminal portion of PrP. Further analysis of prion-infected primary neurons using PrP Sc -specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining

Tanaka

Fujiwara

Suzuki

et al. 2016

Journal of General Virology

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Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines

et al. 2008

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The neuroblastoma-derived cell line N2a is permissive to certain prion strains but resistant sublines unable to accumulate the pathological proteinase-K resistant form of the prion protein can be isolated. We compared for gene expression and phenotypes different N2a sublines that were susceptible or resistant to the 22L prion strain. Karyotypes and comparative genomic hybridization arrays revealed chromosomal imbalances but did not demonstrate a characteristic profile of genomic alterations linked to prion susceptibility. Likewise, we showed that this phenotype was not dependent on the binding of PrPres, the expression of the prion protein gene, or on its primary sequence. We completed this analysis by looking using real-time quantitative PCR at the expression of a set of genes encoding proteins linked to prion biology. None of the candidates could account by itself for the infection phenotype, nevertheless sublines had distinct transcriptional profiles. Taken together, our results do not support a role for specific genomic abnormalities and possible candidate proteins in N2a prion susceptibility. They also reveal genetic heterogeneity among the sublines and serve as a guidance for further investigation into the molecular mechanisms of prion infection.

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Alteration of cell responses to PrPSc in prolonged cell culture and its effect on transmission of PrPSc to neural cells

2012

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The mechanisms and processes of the uptake, intracellular trafficking and intercellular spread of PrP(Sc) and its transfer to neural cells are not clearly defined. The involvement of immune, intestinal, mast or peripheral neural cells in this process also remains unclear. The role of these cell types in the accumulation and transfer of PrP(Sc) to neural cells was investigated following short and prolonged exposure to the Chandler and Obihiro strains of scrapie PrP(Sc) for up to 28 days. Eight cell lines of murine immune, neural, intestinal and fibroblast cell types were tested. After transient degradation phases, certain immune, intestinal and neural cells accumulated PrP(Sc) for up to 28 days postinfection. When co-cultured with N2a-3/EGFP neuroblastoma cells for 4 days followed by several passages, the immune, intestinal and the neural cell lines were able to transfer infection to neural cells. Our results suggest that some of these cell types may have a role in PrP(Sc) accumulation and intercellular spread of PrP(Sc) infection to neural cells in vivo.

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Characterization of Prion Susceptibility in Neuro2a Mouse Neuroblastoma Cell Subclones

Cited by 23 publications

References 43 publications

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining

Comparison of abnormal isoform of prion protein in prion-infected cell lines and primary-cultured neurons by PrPSc-specific immunostaining

Genetic heterogeneity versus molecular analysis of prion susceptibility in neuroblasma N2a sublines

Alteration of cell responses to PrPSc in prolonged cell culture and its effect on transmission of PrPSc to neural cells

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