We have previously described the generation of the Tg(HQK) transgenic mice, in which Dox-induced over-expression of PrPC
specifically in the skeletal muscles causes a primary myopathy that is correlated with accumulation of an N-terminal truncated PrP C1 fragment [6
]. The aim of this study was to determine the molecular basis for the PrP-mediated myopathy by microarray analysis. The ultimate goals are to fully understand the detailed molecular pathways of PrP-mediated myopathy, so that we can better understand the role of PrP in both normal and diseased muscles and provide some clues on the pathogenic mechanism of prion diseases. Utilizing two DNA microarrays, we identified more than 1000 genes that were temporally deregulated in a specific and highly consistent manner following induction of PrPC
over-expression in the muscles of Tg(HQK) mice and the subsequent development of myopathy. The transcriptional profiles in the muscles of Dox-treated Tg(HQK) mice strongly implicate toxicity-induced pro-apoptotic pathways in PrP-mediated myopathy, and they are quite different from the changes previously described in systemic, disuse, and denervation muscle atrophy.
Interestingly, the transcription factor MEF2C was found to be down-regulated at both the mRNA and protein levels in PrPC
-mediated myopathy. MEF2C is expressed specifically in muscle and brain, where it is a target for signaling pathways involving calcium [60
]. MEF2C regulates the expression of a majority of muscle-specific genes, and interacts with members of the MyoD family of proteins to activate muscle differentiation [29
]. Calcium signaling was one of the pathways significantly induced in Dox-treated Tg(HQK) mouse muscles as evidenced by a very small p value of 8.75 × 10-9
. The PrPC
protein has itself been shown to play a role in Ca2+
] and it is possible that over-expression of PrPC
results in perturbations in Ca2+
signaling, which in turn modulates the activity and/or expression of MEF2C. As calcium regulation has also been shown to be altered during prion-induced neurodegeneration, this finding potentially links the molecular changes occurring in Tg(HQK) myopathy to the pathobiology of prion diseases.
The most striking finding is the strong and statistically highly significant induction of a p53-regulated pro-apoptotic network in Tg(HQK) mouse muscles following induction of PrPC
. Central to this network are induction of p53 protein expression and strong induction of genes responsible for arresting the cell cycle, as well as a number of p53-regulated pro-apoptotic (up-regulated) and anti-apoptotic (down-regulated) genes. p53 is a critical tumor suppressor and transcription factor, and it has been linked to cell death in the central nervous system in a number of disorders including most notably neurodegenerative disorders such as Alzheimer's disease and prion diseases [64
]. The expression of p53 protein has been found to rapidly increase in neurons in response to a range of insults including DNA damage, oxidative stress, metabolic compromise, and cellular calcium overload. Over-expression of PrPC
has been shown to enhance staurosporine-induced toxicity and activation of caspase-3 in the HEK293 kidney cell line [67
] and increase sensitivity to apoptotic stimuli via p53-dependent pathways in TSM1 neuronal cell line [20
]. Conversely neurons devoid of PrPC
expression were reported to display lower responsiveness to staurosporine, also via p53-dependent pathways [5
One of the main pro-apoptotic effectors of p53 is BAX, which plays a major role in regulating neuronal death in the brain in response to a number of stimuli [68
]. The role of BAX in prion-induced neurodegeneration is not well understood; both BAX-dependent and BAX-independent mechanisms appear to underlie the action of neurotoxic forms of prion proteins [70
]. However, in the muscle of Dox-treated Tg(HQK) mice, only a marginal increase in BAX expression was observed whereas significant over-expression of other p53 regulated pro-apoptotic proteins, including BAK1, BBC3 and PMAIP1, and MCL1, were detected, suggesting that PrPC
-mediated myopathy observed in this model may depend on Bax-independent pathways that involve BAK1, BBC3, PMAIP1, and MCL1.
We propose a working model to explain the mechanism of PrP-mediated myopathy (Figure ). Dox-induced over-expression of PrPC in the muscles leads to accumulation of the N-terminal truncated PrP C1 fragment, which in turn activates p53, thereby inducing p53-regulated pro-apoptotic networks and myopathic changes.
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. PrPC over-expression also results in down-regulation (more ...)
PrP accumulation has been observed in the skeletal muscles of patients with inclusion-body myositis, polymyositis, dermatomyositis, and neurogenic muscle atrophy, and we have previously reported that over-expression of wild type PrP in the skeletal muscles is sufficient to cause myopathy in the Tg(HQK) mice [[7
] and references therein], which suggest that muscular accumulation of PrP may contribute to the pathogenesis of some human muscle diseases. Our finding that p53-related pathways play a major role in the myopathy in Tg(HQK) mice suggests that p53 and p53-related pathways may also be critical to the pathology of some human muscle disease patients and p53 and p53-related pathways may serve as potential targets for therapeutics development against these muscle diseases.
As we have previously reported [7
], the preferential accumulation of the truncated PrP C1 fragment, which is generated through endoproteolysis of PrPC
during normal protein processing in the brain [8
] and the muscle [7
], was closely correlated with myopathic changes in Dox-treated Tg(HQK) mice. We hypothesize that it is this C1 fragment that is the toxic species in the Tg(HQK) model, which is supported by recent reports showing that over-expression of the C1 fragment increases cell death and caspase-3 activity through a p53-dependent mechanism [20
]. Truncation of PrPC
occurs between residues 110 and 111 within a region shown to play a pivotal role in its conformational transition to PrPSc
. So a better understanding of modulation of this cleavage event and the mechanism for the truncated PrP fragments as mediators of a toxic cellular response may be very important in dissecting prion disease pathogenesis.