Flow Cytometric Detection of PrP^Scin Neurons and Glial Cells from Prion-Infected Mouse Brains

Abstract

In prion diseases, an abnormal isoform of prion protein (PrP) accumulates in neurons, astrocytes, and microglia in the brains of animals affected by prions. Detailed analyses of PrP-positive neurons and glial cells are required to clarify their pathophysiological roles in the disease. Here, we report a novel method for the detection of PrP in neurons and glial cells from the brains of prion-infected mice by flow cytometry using PrP-specific staining with monoclonal antibody (MAb) 132. The combination of PrP staining and immunolabeling of neural cell markers clearly distinguished neurons, astrocytes, and microglia that were positive for PrP from those that were PrP negative. The flow cytometric analysis of PrP revealed the appearance of PrP-positive neurons, astrocytes, and microglia at 60 days after intracerebral prion inoculation, suggesting the presence of PrP in the glial cells, as well as in neurons, from an early stage of infection. Moreover, the kinetic analysis of PrP revealed a continuous increase in the proportion of PrP-positive cells for all cell types with disease progression. Finally, we applied this method to isolate neurons, astrocytes, and microglia positive for PrP from a prion-infected mouse brain by florescence-activated cell sorting. The method described here enables comprehensive analyses specific to PrP-positive neurons, astrocytes, and microglia that will contribute to the understanding of the pathophysiological roles of neurons and glial cells in PrP-associated pathogenesis. Although formation of PrP in neurons is associated closely with neurodegeneration in prion diseases, the mechanism of neurodegeneration is not understood completely. On the other hand, recent studies proposed the important roles of glial cells in PrP-associated pathogenesis, such as the intracerebral spread of PrP and clearance of PrP from the brain. Despite the great need for detailed analyses of PrP-positive neurons and glial cells, methods available for cell type-specific analysis of PrP have been limited thus far to microscopic observations. Here, we have established a novel high-throughput method for flow cytometric detection of PrP in cells with more accurate quantitative performance. By applying this method, we succeeded in isolating PrP-positive cells from the prion-infected mouse brains via fluorescence-activated cell sorting. This allows us to perform further detailed analysis specific to PrP-positive neurons and glial cells for the clarification of pathological changes in neurons and pathophysiological roles of glial cells.

Microglia in neurodegeneration

“…Prion-related neurodegeneration includes neuronal loss, increased proinflammatory microglia, and spongiform changes 90 . Microglia phagocytose PrP sc as early as 60 days postinfection 91 , and their depletion increases prion titers and susceptibility to prion infection 92 , suggesting they help control prion disease.…”

Enhancement of binding avidity by bivalent binding enables PrPSc-specific detection by anti-PrP monoclonal antibody 132

“…Thus, weak monovalent binding of mAb 132 to monomeric PrP C diminishes the signals from PrP C to background level, whereas after exposure of the epitope, mAb 132 selectively binds to oligomeric PrP Sc in a bivalent manner (Figs 8, 9A and 9D,). This combination provides a better signal-noise-ratio and therefore enables reliable PrP Sc detection in cells and sections using IFA, cell-based ELISA, and flow cytometry (Fig 8) [18,20,21,32]. However, considering the mechanism of PrP Sc -specific detection of mAb 132 described above, mAb 132 will react with PrP C if more than two PrP C molecules exist within the distance that allows bivalent binding of IgG (Fig 9B).…”

“…The immunofluorescence staining of frozen sections was performed as described elsewhere [18,21] with minor modifications. Briefly, the frozen brain sections were fixed and permeabilized as described above.…”

“…However, since most pan-PrP mAbs react with both PrP isoforms, manipulation of the detector gain or exposure time is required to set a threshold level just above the PrP C signals. Recently, we reported that the mAb 132 recognizing mouse PrP amino acids (aa) 119–127, is useful for reliable PrP Sc -specific staining in cells or frozen tissue sections by IFA without PK treatment [18–20] and flow cytometry [21], even though this mAb is classified as a group of pan-PrP antibodies that is incapable of distinguishing PrP Sc from PrP C . We also reported that mAb 132 detects both PrP Sc -sen and PrP Sc -res if not all [22].…”

Microglial Turnover in Ageing-Related Neurodegeneration: Therapeutic Avenue to Intervene in Disease Progression

“…Prion-related neurodegeneration includes increased neuronal loss and proinflammatory microglia. Microglia and other CNS macrophages phagocytose PrP Sc approximately 60 days earlier after sensing infection [ 109 ], but their depletion increases prion infection susceptibility [ 110 ], suggesting that microglia play a major role in controlling prion disease. The double-edged sword metaphor applies here as well for microglia because they produce ROS in response to the PrP106-126 fragment and promote PrP-induced neurotoxicity.…”