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Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie

View Article: PubMed Central

ABSTRACT

Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system.

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Cellular localization of PrPd in cerebellar slices. 9 weeks p.i., Mock exposed or 22L infected slices were subjected to antigen denaturation and immunofluorescence staining. Cells in the molecular layer of folium IX were analyzed by confocal microscopy using digital zoom with identical imaging settings for each sample group. PrPd was detected with mAb 4H11 (green). (A) Neurons were stained for β-3-tubulin (red). The insets respresent magnifications of the indicated region. (B) Purkinje cells were stained with mAb anti-calbindin (red). (C) Astrocytes were labeled with pAb anti-GFAP (red). Arrows mark co-localization of PrPd with lamp-1 within astrocytes. (D) PAb anti-iba-1 was used to detect microglia (red). Arrowheads mark degenerated nuclei within microglia, arrows mark lysosomes. Lysosomes were labeled with mAb anti-lamp-1 (purple) and nuclei were counterstained with Hoechst. Mock exposed slices served as controls. Scale bar: 5 μm.
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Figure 6: Cellular localization of PrPd in cerebellar slices. 9 weeks p.i., Mock exposed or 22L infected slices were subjected to antigen denaturation and immunofluorescence staining. Cells in the molecular layer of folium IX were analyzed by confocal microscopy using digital zoom with identical imaging settings for each sample group. PrPd was detected with mAb 4H11 (green). (A) Neurons were stained for β-3-tubulin (red). The insets respresent magnifications of the indicated region. (B) Purkinje cells were stained with mAb anti-calbindin (red). (C) Astrocytes were labeled with pAb anti-GFAP (red). Arrows mark co-localization of PrPd with lamp-1 within astrocytes. (D) PAb anti-iba-1 was used to detect microglia (red). Arrowheads mark degenerated nuclei within microglia, arrows mark lysosomes. Lysosomes were labeled with mAb anti-lamp-1 (purple) and nuclei were counterstained with Hoechst. Mock exposed slices served as controls. Scale bar: 5 μm.

Mentions: To identify the cellular localization of PrPd, double labeling of PrPd with neurons (β-3-tubulin), Purkinje cells (calbindin), astrocytes (GFAP), and microglia (iba-1) was performed. Potential subcellular deposition of PrPd within lysosomes was assessed using mAb targeting lamp-1. The molecular and Purkinje cell layers exhibited intense spot-like to fibrillar PrPd deposits (Figure 6). Spot-like PrPd deposition was observed in areas with β-3-tubulin staining (Figure 6A). In some cases, PrPd colocalized with neuronal lysosomes (Figure 6A, insets). Purkinje cells were surrounded by cells with intense PrPd staining, while Purkinje cell bodies showed no or only very faint staining for PrPd (Figure 6B; Supplementary Figure 2). Interestingly, PrPd deposition was found extracellularly between Purkinje cells in close vicinity (Supplementary Figure 2). Surrounding cells stained positive for glial fibrillar acidic protein (GFAP), suggesting that they represent Bergmann glia (Figure 6C). Here, PrPd at least partially colocalized with lamp-1, indicative of lysosomal localization of PrPd within astrocytes (Figure 6C, arrows). PrPd deposition in astrocytes has been reported previously for 22L infected C57BL/6 mice (Diedrich et al., 1991). PrPd staining was also associated with microglia (Figure 6D). In some instances, degenerated nuclei were identified in iba-1 positive microglia in the 22L infected cerebellar slices (Figure 6D, arrowheads). In conclusion, in 22L infected cerebellar slices from C57BL/6JRj mice, PrPd deposits as spot-like or fibrillar structures in the neuropil and is associated with astrocytes, microglia and neurons. In line with in vivo studies (Moore et al., 2008; Sarasa et al., 2012), PrPd is not (or only at very low levels) located in Purkinje cells.


Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie
Cellular localization of PrPd in cerebellar slices. 9 weeks p.i., Mock exposed or 22L infected slices were subjected to antigen denaturation and immunofluorescence staining. Cells in the molecular layer of folium IX were analyzed by confocal microscopy using digital zoom with identical imaging settings for each sample group. PrPd was detected with mAb 4H11 (green). (A) Neurons were stained for β-3-tubulin (red). The insets respresent magnifications of the indicated region. (B) Purkinje cells were stained with mAb anti-calbindin (red). (C) Astrocytes were labeled with pAb anti-GFAP (red). Arrows mark co-localization of PrPd with lamp-1 within astrocytes. (D) PAb anti-iba-1 was used to detect microglia (red). Arrowheads mark degenerated nuclei within microglia, arrows mark lysosomes. Lysosomes were labeled with mAb anti-lamp-1 (purple) and nuclei were counterstained with Hoechst. Mock exposed slices served as controls. Scale bar: 5 μm.
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Figure 6: Cellular localization of PrPd in cerebellar slices. 9 weeks p.i., Mock exposed or 22L infected slices were subjected to antigen denaturation and immunofluorescence staining. Cells in the molecular layer of folium IX were analyzed by confocal microscopy using digital zoom with identical imaging settings for each sample group. PrPd was detected with mAb 4H11 (green). (A) Neurons were stained for β-3-tubulin (red). The insets respresent magnifications of the indicated region. (B) Purkinje cells were stained with mAb anti-calbindin (red). (C) Astrocytes were labeled with pAb anti-GFAP (red). Arrows mark co-localization of PrPd with lamp-1 within astrocytes. (D) PAb anti-iba-1 was used to detect microglia (red). Arrowheads mark degenerated nuclei within microglia, arrows mark lysosomes. Lysosomes were labeled with mAb anti-lamp-1 (purple) and nuclei were counterstained with Hoechst. Mock exposed slices served as controls. Scale bar: 5 μm.
Mentions: To identify the cellular localization of PrPd, double labeling of PrPd with neurons (β-3-tubulin), Purkinje cells (calbindin), astrocytes (GFAP), and microglia (iba-1) was performed. Potential subcellular deposition of PrPd within lysosomes was assessed using mAb targeting lamp-1. The molecular and Purkinje cell layers exhibited intense spot-like to fibrillar PrPd deposits (Figure 6). Spot-like PrPd deposition was observed in areas with β-3-tubulin staining (Figure 6A). In some cases, PrPd colocalized with neuronal lysosomes (Figure 6A, insets). Purkinje cells were surrounded by cells with intense PrPd staining, while Purkinje cell bodies showed no or only very faint staining for PrPd (Figure 6B; Supplementary Figure 2). Interestingly, PrPd deposition was found extracellularly between Purkinje cells in close vicinity (Supplementary Figure 2). Surrounding cells stained positive for glial fibrillar acidic protein (GFAP), suggesting that they represent Bergmann glia (Figure 6C). Here, PrPd at least partially colocalized with lamp-1, indicative of lysosomal localization of PrPd within astrocytes (Figure 6C, arrows). PrPd deposition in astrocytes has been reported previously for 22L infected C57BL/6 mice (Diedrich et al., 1991). PrPd staining was also associated with microglia (Figure 6D). In some instances, degenerated nuclei were identified in iba-1 positive microglia in the 22L infected cerebellar slices (Figure 6D, arrowheads). In conclusion, in 22L infected cerebellar slices from C57BL/6JRj mice, PrPd deposits as spot-like or fibrillar structures in the neuropil and is associated with astrocytes, microglia and neurons. In line with in vivo studies (Moore et al., 2008; Sarasa et al., 2012), PrPd is not (or only at very low levels) located in Purkinje cells.

View Article: PubMed Central

ABSTRACT

Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system.

No MeSH data available.


Related in: MedlinePlus