Activation of p53-regulated pro-apoptotic signaling pathways in PrP-mediated myopathy

Abstract

Background: We have reported that doxycycline-induced over-expression of wild type prion protein (PrP) in skeletal muscles of Tg(HQK) mice is sufficient to cause a primary myopathy with no signs of peripheral neuropathy. The preferential accumulation of the truncated PrP C1 fragment was closely correlated with these myopathic changes. In this study we use gene expression profiling to explore the temporal program of molecular changes underlying the PrP-mediated myopathy.

Results: We used DNA microarrays, and confirmatory real-time PCR and Western blot analysis to demonstrate deregulation of a large number of genes in the course of the progressive myopathy in the skeletal muscles of doxycycline-treated Tg(HQK) mice. These include the down-regulation of genes coding for the myofibrillar proteins and transcription factor MEF2c, and up-regulation of genes for lysosomal proteins that is concomitant with increased lysosomal activity in the skeletal muscles. Significantly, there was prominent up-regulation of p53 and p53-regulated genes involved in cell cycle arrest and promotion of apoptosis that paralleled the initiation and progression of the muscle pathology.

Conclusion: The data provides the first in vivo evidence that directly links p53 to a wild type PrP-mediated disease. It is evident that several mechanistic features contribute to the myopathy observed in PrP over-expressing mice and that p53-related apoptotic pathways appear to play a major role.

Figure 1

Clustering of gene expression data. A. Measurements of relative gene expression for 6 time points (after 0, 4, 7, 14, 30, 60 days of Dox treatment) in Tg(HQK) mice (HQK) and PrP knockout mice (KO). Mice were treated with 6 g Dox/kg food, and three animals were taken at each time point as indicated. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected to microarray analysis, yielding expression profiles of genes with normalized expression ratios. Red and purple represent relative over-expression and under-expression, respectively, and the color intensity represents the magnitude of digression. B. Bioinformatic analysis (Ingenuity Pathways Analysis) to determine the top biological functions and associated p values of the selected genes is shown. The top four categories are listed for diseases and disorders, molecular and cellular functions, physiological system and development and function, and pathways associated with toxicity.

Figure 2

MEF2C protein level is down-regulated in the skeletal muscles of Tg(HQK) mice treated with doxycycline (Dox). Tg(HQK) mice were treated with 6 g Dox/kg food for 0–60 days as indicated, and three animals were taken at each time point. Skeletal muscles (quadriceps) from the hind legs were subjected to immunoblot analysis in three blots. Fifteen micrograms of total proteins was loaded for each sample. Skeletal muscle (quadriceps) sample from an untreated wild type FVB mouse (WT) serves as the control to normalize data from the triplicate blots. A. A representative immunoblot probed with anti-MEF2C antibody followed by probing with an anti-actin antibody after stripping. B. Plot of the MEF2C protein levels over increasing duration of Dox treatment. The MEF2C protein level for each sample was normalized against the actin level in each blot and expressed as the ratio against the normalized MEF2C protein level in the untreated wild type FVB mouse on the same blot. The error bars denote standard errors calculated from the three blots. The bars with asterisk(s) indicate a statistically significant difference when compared to the 0 day Tg(HQK) samples. *p < 0.05; ** p < 0.001.

Figure 3

Real-time PCR analysis of Atrogin-1 and MuRF1. qRT-PCR analysis of Atrogin-1 (A) and MuRF1 (B) gene expression in RNA samples from Tg(HQK) mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 4 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food beginning on day 0. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. * p < 0.01; ** p < 0.001.

Figure 4

Real-time PCR analysis of mdm2 and p53. qRT-PCR analysis of mdm2 (grey) and p53 (black) gene expression in RNA samples from Tg(HQK) PrP over-expressing mice relative to similarly treated wild-type control mice. Measurements of relative gene expression for 5 time points (over 4–60 days) in mice following treatment with 6 g Dox/kg food. Total RNA was extracted from skeletal muscles (quadriceps) from the hind legs and subjected qRT-PCR analysis. Results represent the mean ± s.e.m. of triplicate measurements performed. ** p < 0.01; *** p < 0.001.

Figure 5

Total p53 protein level is up-regulated in the skeletal muscles of Tg(HQK) mice treated with doxycycline. Tg(HQK) mice were treated with 6 g Dox/kg food for 0–60 days as indicated, and three animals were taken at each time point. Skeletal muscles (quadriceps) from the hind legs were subjected to immunoblot analysis in three blots. Twenty micrograms of total proteins was loaded for each sample. Skeletal muscle (quadriceps) sample from an untreated wild type FVB mouse (WT) serves as the control to normalize data from the triplicate blots. A. A representative immunoblot probed with anti-p53 antibody followed by probing with an anti-actin antibody after stripping. B. Plot of the total p53 protein levels over increasing duration of Dox treatment. The p53 protein level for each sample was normalized against the actin level in each blot and expressed as the ratio against the normalized total p53 protein level in the untreated wild type FVB mouse on the same blot. The error bars denote standard errors calculated from the three blots. The bars with asterisk(s) indicate a statistically significant difference when compared to the 0 day Tg(HQK) samples. *p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 6

p53-regulated pathway analysis using the Ingenuity Pathway Knowledge Base (IPKB). This figure illustrates potential functional relationships of TP53 responsive genes de-regulated in the muscles of Dox-treated Tg(HQK) mice. Direct (solid lines) and indirect (dashed lines) interactions reported for these genes (grey shading) in the IPKB database. Color shading corresponds to the type of de-regulation: red for up-regulated genes, and green for down-regulated genes. The shape of the node indicates the major function of the protein (see key), and a line denotes binding of the products of the two genes while a line with an arrow denotes 'acts on'.

Figure 7

Mechanism of PrP-mediated myopathy. Accumulation of an N-terminal truncated PrP C1 fragment in muscle activates p53 resulting in the induction of p53-regulated pro-apoptotic networks and myopathic changes. PrP^C over-expression also results in down-regulation of MEF2C, which may be partially responsible for the progressive central nuclei localization observed in the muscles of Dox-treated Tg(HQK) mice.