Prion diseases are a protein-based infectious diseases of the nervous system that result in progressive and extensive neurodegeneration throughout the brain. The mechanisms leading to neurodegeneration are incompletely understood, but loss of function of PrionC, gain-of-toxic function of PrionSc, activation of cellular and in particular ER stress, neuronal death pathways such as autophagy and apoptosis, and chronic brain inflammation all appear to play a role. Experimental in vitro or in vivo approaches typically investigate one of the potential pathogenic processes at a time, and often quite elegantly demonstrate their relevance in the disease process. However, to investigate which of these processes is the most pathogenic and contributes the most to the disease process, conventional experimental approaches are more limited. To address this issue, we therefore used a network analysis approach and looked for a set of genes determining the stability of the network using a Boolean dynamical system. By identifying a set of core regulatory genes, most of which are part of the immune response, that can lock a larger gene network into a stable diseased state, we found that it is the process of brain inflammation that is the most likely to play the major pathogenic role in prion disease.
Despite the fact that six of the 333 DEGs described by Hwang et al. are related to the oxidative stress response, only one of them is included in our global network (PRDX6) and none in the core regulatory network. In neurons, the accumulation of misfolded proteins also leads to endoplasmic reticulum (ER) stress. The normal unfolded protein response (UPR), which is a consequence of the ER stress, should function to clear the misfolded proteins. But it is thought that pro-inflammatory cytokines released by microglia lead to an upregulation of ER stress and an atypical form of UPR in neurons
[15–17]. Furthermore, it has been hypothesized that prolonged ER stress activates cell death pathways
[17, 18]. Cellular stress is one of the possible mechanisms triggering neurodegeneration. Necrosis in acute brain injury is a result of the release of nitric oxide, reactive oxygen species, calcium and glutamate
. The role of prion in triggering the stress response is still not understood, but a toxic effect caused by free radicals produced due to an imbalance in the intracellular cupper levels has been previously associated with prion disease and other neurodegenerative diseases
[19, 20]. Whether this imbalance is due to the loss of function (lack of prion directly or indirectly involved in dismutase activity) or the gain of new functions (aggregation of PrPSc) is still unclear, but there is evidence that the toxicity results from prion dependent processes (alteration of PrP-mediated signaling, PrP mislocalization and oligomerization) in murine models of scrapie
The Ccaat-enhance binding protein (CEBP) transcription factor family has been implicated in the differentiation of myelomonocytic cells and the regulation of gene expression during the activation of macrophages
. The isoform CEBPA regulates the expression of antioxidant proteins. CEBPA-deficient mice were found to be susceptible to hyperoxic conditions and prion was one of the DEGs identified, thus suggesting a role of wild type prion in lung injury
. Overexpression of the CEBPD isoform is observed in Alzheimer’s
 and in prion disease
. Colony-stimulating factor 1 (CSF1) and its receptor CSF1R are involved in the process of macrophage DNA synthesis, which is induced by the presence of amyloid beta and prion protein. The presence of misfolded amyloid beta precursor and prion protein promotes macrophage survival in bone marrow cells
. Integrin beta 2 is encoded by ITGB2 and is known to selectively bind to neuronal cells damaged by cleaved prion. It is also involved in neuronal cell killing by activated microglial cells
. Transglutaminase 2 is encoded by the TGM2 gene and may contribute to the cross-linking of soluble peptides preventing amyloid formation of alpha-synuclein or prion protein, thus increasing concentrations of soluble toxic oligomers
. Overexpression of TGM2 is also observed in Huntington and Alzheimer’s diseases
. CX3CR1 is part of the complement system and encodes the receptor for the chemokine fractalkine. Antagonists of chemokine receptors have been shown to play a neuroprotective role
 in several neurodegenerative diseases including prion disease. Also, thiazolopyrimidine derivatives are antagonists of CX3CR1 and have been developed as drugs for prion disease therapy
. TGFB1 is a cytokine which is overexpressed in prion disease. Signaling through the TGFB1 receptor is potentially responsible for the chronic inflammatory processes by regulating an atypical microglia phenotype
. LGALS3 encodes galectin-3, a beta-galactoside binding lectin. It has been shown that lectin binding and normal prion (PrPC) glycosylation are altered during aging
. STAT1 transcription factor signaling is increased in mouse models lacking PrPC, indicating a neuroprotective role of PrPC[34, 35]. The STAT3 transcription factor is important for the anti-apoptotic activity of humanin and therefore for humanin-mediated neuroprotection after Alzheimer disease-related insult
. The protective role of humanin was also observed in prion disease
The possible mechanistic links of the remaining genes in the SCC (TLR2, NCF1, PTPN6 and B2M) to prion disease are still open. Thus, these genes represent a new set with a potential crucial function in prion disease pathogenesis.
The gene product of TLR2, or Toll-like-receptor 2, is a pattern recognition receptor found primarily on innate immunity cells including microglia/macrophages
[38, 39]). Although toll-like receptors are known to recognize misfolded proteins and trigger stress responses in various neurodegenerative diseases, TLR2 has not been directly linked with the recognition of prion deposition
. Interestingly, pharmacological inhibitors of Toll-like-receptors, including TLR2, are being studied for the treatment of various inflammatory diseases
). The gene product of NCF1, or Neutrophil-cytosolic-factor 1, is a component of the neutrophil NADPH, and enzyme complex that produces a superoxide anion, a reactive radical
. The gene product of PTPN6, also called SHP-1 (Src homology region 2 domain-containing phosphatase-1), codes for Tyrosine-protein-phosphatase non-receptor type 6, a protein tyrosine phosphatase involved in cell differentiation, in particular hematopoetic cells
. B2M codes for β2 microglobulin, a component of MHC class I molecules, and its expression in neurons is regulated during injury
. These factors point again to a major involvement of the innate immune system in the pathogenesis of prion disease, and furthermore suggest the existence of new targets for possible therapeutic intervention.
In another transcriptomics study by Sorensen et al., C57BL/6 or VM mice were inoculated intracerebrally with three strains of scrapie prion that differed from those used in the study by Hwang et al. Seventeen genes were found to be related to inflammation and microglia activation. Ten of these genes were also among the 333 DEG in the study by Hwang et al.: six are located in our core network (ABCA1, CLU, TF and SOX9) and two in our SCC (B2M and TGFB1). However, it should be noted that CLU was placed in the cell death category and TF into the iron homeostasis category by Hwang et al. All genes were similarly upregulated in both studies. We decided to follow up with the network analysis of data from the latter publication since it was based on larger number of mice model -prion strain combinations and therefore provided more comprehensive list of DEGs. Although, both Hwang’s and Sorensen’s studies included analysis aimed on identifying genes related to inflammatory processes, a stability and dynamics analysis of the gene regulatory network was performed for the first time in the present study, allowing for the identification of brain inflammation as the main prion disease process among all the possible others.
Inflammation response could be an overrepresented functional category in a great number of diseases. In the particular case of neurodegenerative diseases belonging to the class of protein misfolding diseases, it is well established that neuroinflammation plays a role. What we consider remarkable and constitutes our main finding is the key role that neuroinflammation plays in the specific case of prion disease, connecting different functional modules and constituting a switch that allows the network to reach a self-maintained disease state, once triggering factors (protein deposition and the formation of amyloid plaques) initiate the process. According to our simulations, the special topology that connects neuroinflammation elements (a cluster of positive feed-back loops or SCC) makes the regulatory core sensitive under perturbation to easily transit from inactive (healthy) to active (diseased) states but become very stable once the active state is reached.
To experimentally test the role of the genes of the identified master regulatory core in prion disease, we envisage inoculating PrionSc into knockout mice that are lacking one of the 16 SCC genes. Subsequently, the mice would be analyzed pathologically with regard to neuronal function and death, as well as for DEGs for which the network analysis would be repeated. This analysis would be particularly interesting for the factors whose explicit role has never been demonstrated in prion pathogenesis (TLR2, NCF1, PTPN6).