Manganese upregulates cellular prion protein and contributes to altered stabilization and proteolysis: relevance to role of metals in pathogenesis of prion disease

Abstract

Prion diseases are fatal neurodegenerative diseases resulting from misfolding of normal cellular prion (PrP(C)) into an abnormal form of scrapie prion (PrP(Sc)). The cellular mechanisms underlying the misfolding of PrP(C) are not well understood. Since cellular prion proteins harbor divalent metal-binding sites in the N-terminal region, we examined the effect of manganese on PrP(C) processing in in vitro models of prion disease. Exposure to manganese significantly increased PrP(C) levels both in cytosolic and in membrane-rich fractions in a time-dependent manner. Manganese-induced PrP(C) upregulation was independent of messenger RNA transcription or stability. Additionally, manganese treatment did not alter the PrP(C) degradation by either proteasomal or lysosomal pathways. Interestingly, pulse-chase analysis showed that the PrP(C) turnover rate was significantly altered with manganese treatment, indicating increased stability of PrP(C) with the metal exposure. Limited proteolysis studies with proteinase-K further supported that manganese increases the stability of PrP(C). Incubation of mouse brain slice cultures with manganese also resulted in increased prion protein levels and higher intracellular manganese accumulation. Furthermore, exposure of manganese to an infectious prion cell model, mouse Rocky Mountain Laboratory-infected CAD5 cells, significantly increased prion protein levels. Collectively, our results demonstrate for the first time that divalent metal manganese can alter the stability of prion proteins and suggest that manganese-induced stabilization of prion protein may play a role in prion protein misfolding and prion disease pathogenesis.

FIG. 1.

Manganese-induced cellular prion protein (PrP^C) upregulation in mouse neuronal cells. (A) Western blot analysis of PrP^C at various time points following manganese treatment. The cells were treated with manganese (100μM) for 0, 6, 12, 18, and 24 h and harvested. Cytosolic fractions and membrane-rich fractions were obtained and analyzed by Western blotting. (B) Densitometry analysis of PrP^C bands (17–37 kDa) in (A). *p < 0.05 or **p < 0.01 compared with the control group. (C) ICC expression of PrP in cells with and without manganese treatment. PrP^C staining is shown in green and nucleus staining is in blue.

FIG. 2.

Effect of manganese on mRNA levels, ubiquitin proteasomal system, and lysozyme activity. (A) Comparison of PrP mRNA levels in manganese-treated and control mouse neuronal cells. The cells were treated with 100μM manganese for 24 h, and PrP mRNA levels were determined by quantitative reverse-transcriptase polymerase chain reaction. (B) Determination of PrP mRNA stability with manganese treatment. The cells were treated with actinomycin D to inhibit transcription of mRNA, and PrP mRNA levels were determined at 3, 6, and 12 h in manganese-treated and control cells. (C) Measurement of proteasomal activity with manganese treatment. Chymotrypsin-like activity was measured at 24 h following manganese treatment. (D) Evaluation of high–molecular weight ubiquitinated protein formation with manganese treatment. The cells were treated with manganese for up to 24 h; ubiquitinated proteins were measured in soluble and insoluble fractions by Western blot at 0, 6, 12, 18, and 24 h. A known proteasome inhibitor MG132 was used as a positive control. (E) Measurement of lysosomal activity with manganese treatment. Lysosomal activity was measured following 100μM manganese treatment at 24 h. Egg white lysozyme was used as a positive control. Experiments were repeated three to four times in each assay.

FIG. 3.

Effect of manganese on PrP^C turnover rate measured by pulse-chase analysis. (A) Confluent mouse neuronal cells were metabolically labeled with 300 μCi/ml [³⁵S]methionine for 1 h at 37°C. After the pulse, cells were incubated in cell culture medium without [³⁵S]methionine in the presence or absence of 100μM manganese at 37°C for the indicated chase time periods. PrP^C was immunoprecipitated with 3F4 antibody and then subjected to SDS-PAGE and autoradiography. (B) Densitometric evaluation of autoradiogram. PrP^C collected 1 h after the radioactive pulse was set as 100% PrP^C population. Decreases in protein amounts following the chase periods are expressed as a percentage of total protein plotted as a function of time. The data points were fitted to an exponential curve using nonlinear regression analysis. Each data point represents the mean ± SEM for three replicates.

FIG. 4.

Manganese decreases PK-dependent PrP^C proteolysis. (A) Limited proteolysis of PrP^C in presence or absence of manganese. The cells treated with 100μM manganese and lysates were prepared as described in the “Materials and Methods” section. Samples were separated into equal fractions and treated with 1 μg/ml PK at 37°C. PK digestion was inhibited at indicated time periods with addition of 4mM PMSF and processed for Western blotting with 3F4 antibody or β-actin antibody; (B) Densitometric analysis of the PrP^C and β-actin bands was quantified and fit to a single-phase exponential decay. Each data point represents mean ± SEM from three individual experiments performed.

FIG. 5.

Manganese-induced PrP^C upregulation in mouse brain slices. Mouse brain slice cultures were exposed to 300μM manganese for 1, 3, and 7 days, and slices were then homogenized and subjected to Western blot and metal analysis. (A) Representative Western blot analysis of total PrP^C levels in mouse brain slices treated with manganese and in untreated slices is shown at top. (B) Below, the representative Western blot image is the quantification of band intensity of PrP^C normalized with β-actin levels. Each data point represents experiments performed in triplicate *p < 0.05.

FIG. 6.

Divalent cation levels in mouse brain slices following manganese treatment. Quantitative analysis of divalent cation levels in mouse brain slice culture treated with 300μM manganese for 1, 3, and 7 days is shown in (A) Manganese, (B) Copper, and (C) Iron. Each data point represents experiments normalized to wet weight of mouse brain slices performed in triplicate ***p < 0.001.

FIG. 7.

Manganese-induced PrP^C upregulation in an infectious cell culture model of prion disease. (A) PK-resistant PrP^Sc levels in RML-infected CAD5 cells. (B) Western blot analysis of PrP^C at 12 h with 200μM manganese treatment in both uninfected and RML-infected CAD5 cells. (C) Densitometry analysis of PrP^C bands (17–37 kDa) in (A).